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Kranc W, Kaczmarek M, Kowalska K, Pieńkowski W, Ciesiółka S, Konwerska A, Mozdziak P, Brązert M, Jeseta M, Spaczyński RZ, Pawelczyk L, Kempisty B. Morphological characteristics, extracellular vesicle structure and stem-like specificity of human follicular fluid cell subpopulation during osteodifferentiation. Exp Mol Pathol 2025; 142:104965. [PMID: 40253818 DOI: 10.1016/j.yexmp.2025.104965] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2024] [Revised: 03/28/2025] [Accepted: 04/03/2025] [Indexed: 04/22/2025]
Abstract
Extracellular vesicles can play an important role in the processes occurring after stem cell transplantation, preventing cell apoptosis, stimulating immunological processes, and promoting the synthesis of extracellular matrix. Human follicular fluid (FF) can be a source of a subpopulation of cells with mesenchymal stem cells (MSCs) properties. Moreover these subpopulations of FF cells can differentiate into osteoblasts. In presented studies flow cytometry of ovarian FF cells confirmed positive expression of MSCs markers such as: CD44, CD90, CD105, CD73 and negative expression of a hematopoietic marker: CD45. The CD90+, CD105+, CD45- cell subpopulation has been obtained during magnetic separation using appropriate antibodies conjugated with microbeads. The extracellular vesicles (EVs) secreted by the cells during osteodifferentiation process differed from those secreted by cells culture in the basal medium. Based on the previous and current electron microscopy research, changes in size, number, and shape would support the notion that released EVs could be crucial to the ovarian FF cell subpopulation differentiation process. Osteogenic differentiation has been confirmed via Alizarin red staining. Therefore, follicular fluid (FF) can be a new source of a cell subpopulation with MSC properties, with the cells capable of differentiating into the osteogenic lineage. EVs could play a key role as mediators in tissue regeneration, especially bone tissue regeneration.
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Affiliation(s)
- Wiesława Kranc
- Department of Anatomy, Poznan University of Medical Sciences, 6 Święcickiego St., 60-781 Poznan, Poland.
| | - Mariusz Kaczmarek
- Department of Cancer Diagnostics and Immunology, Greater Poland Cancer Center, 15 Garbary St., 61-866 Poznań, Poland; Department of Cancer Immunology, Poznan University of Medical Sciences, 5 Garbary St., 61-866 Poznań, Poland.
| | - Katarzyna Kowalska
- Department of Histology and Embryology, Poznan University of Medical Sciences, 6 Swiecickiego St., 60-781 Poznan, Poland.
| | - Wojciech Pieńkowski
- Division of Perinatology and Women's Diseases, Poznan University of Medical Sciences, 33 Polna St. 60-535 Poznan, Poland.
| | - Sylwia Ciesiółka
- Department of Histology and Embryology, Poznan University of Medical Sciences, 6 Swiecickiego St., 60-781 Poznan, Poland.
| | - Aneta Konwerska
- Department of Histology and Embryology, Poznan University of Medical Sciences, 6 Swiecickiego St., 60-781 Poznan, Poland.
| | - Paul Mozdziak
- Prestage Department of Poultry Sciences, North Carolina State University, Raleigh, NC 27695, USA; Physiology Graduate Faculty, North Carolina State University, Raleigh, NC 27695, USA.
| | - Maciej Brązert
- Department of Diagnostic and Treatment of Infertility, Department of Gynecological Endocrinology and Infertility Treatment Karol Marcinkowski University, Poznan University of Medical Sciences, 33 Polna St., 60-535 Poznan, Poland.
| | - Michal Jeseta
- Department of Obstetrics and Gynecology, University Hospital and Masaryk University, 62500 Brno, Czechia.
| | - Robert Z Spaczyński
- Center for Gynecology, Obstetrics and Infertility Treatment Pastelova, Pastelowa 8, 60-198, Poznan, Poland..
| | - Leszek Pawelczyk
- Department of Diagnostic and Treatment of Infertility, Department of Gynecological Endocrinology and Infertility Treatment Karol Marcinkowski University, Poznan University of Medical Sciences, 33 Polna St., 60-535 Poznan, Poland.
| | - Bartosz Kempisty
- Physiology Graduate Faculty, North Carolina State University, Raleigh, NC 27695, USA; Department of Obstetrics and Gynecology, University Hospital and Masaryk University, 62500 Brno, Czechia; Division of Anatomy, Department of Human Morphology and Embryology, Faculty of Medicine, Wroclaw Medical University, Chalubinskiego 6a, 50-368 Wroclaw, Poland; Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Torun, 1 Lwowska St., 87-100 Torun, Poland.
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Riva F, Bloise N, Omes C, Ceccarelli G, Fassina L, Nappi RE, Visai L. Human Ovarian Follicular Fluid Mesenchymal Stem Cells Express Osteogenic Markers When Cultured on Bioglass 58S-Coated Titanium Scaffolds. MATERIALS (BASEL, SWITZERLAND) 2023; 16:ma16103676. [PMID: 37241304 DOI: 10.3390/ma16103676] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/30/2023] [Revised: 04/28/2023] [Accepted: 05/08/2023] [Indexed: 05/28/2023]
Abstract
Recent studies have reported that stem cells (human follicular fluid mesenchymal stem cells or hFF-MSCs) are present in ovarian follicular fluid (hFF) and that they have a proliferative and differentiative potential which is similar to that of MSCs derived from other adult tissue. These mesenchymal stem cells, isolated from human follicular fluid waste matter discarded after retrieval of oocytes during the IVF process, constitute another, as yet unutilized, source of stem cell materials. There has been little work on the compatibility of these hFF-MSCs with scaffolds useful for bone tissue engineering applications and the aim of this study was to evaluate the osteogenic capacity of hFF-MSCs seeded on bioglass 58S-coated titanium and to provide an assessment of their suitability for bone tissue engineering purposes. Following a chemical and morphological characterization with scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), cell viability, morphology and expression of specific osteogenic markers were examined after 7 and 21 days of culture. The hFF-MSCs seeded on bioglass and cultured with osteogenic factors, when compared with those seeded on tissue culture plate or on uncoated titanium, exhibited enhanced cell viability and osteogenic differentiation, as reflected by increased calcium deposition and increased ALP activity with expression and production of bone-related proteins. Taken together, these results demonstrate that MSCs from human follicular fluid waste materials can be easily cultured in titanium scaffolds coated with bioglass, having osteoinductive properties. This process has significant potential for regenerative medicine applications and indicates that hFF-MSCs may be a valid alternative to hBM-MSC cells in experimental models in bone tissue engineering.
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Affiliation(s)
- Federica Riva
- Histology and Embryology Unit, Department of Public Health, Experimental and Forensic Medicine, University of Pavia, 27100 Pavia, Italy
| | - Nora Bloise
- Department of Molecular Medicine, Centre for Health Technologies (CHT), INSTM UdR of Pavia, University of Pavia, 27100 Pavia, Italy
- Medicina Clinica-Specialistica, UOR5 Laboratorio di Nanotecnologie, ICS Maugeri, IRCCS, 27100 Pavia, Italy
| | - Claudia Omes
- Center for Reproductive Medicine, Obstetrics and Gynecology Unit 2, Woman and Child Health Department, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
| | - Gabriele Ceccarelli
- Human Anatomy Unit, Department of Public Health, Experimental and Forensic Medicine, Centre for Health Technologies (CHT), University of Pavia, 27100 Pavia, Italy
| | - Lorenzo Fassina
- Department of Electrical, Computer and Biomedical Engineering, Centre for Health Technologies (CHT), University of Pavia, 27100 Pavia, Italy
| | - Rossella Elena Nappi
- Center for Reproductive Medicine, Obstetrics and Gynecology Unit 2, Woman and Child Health Department, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy
- Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, University of Pavia, 27100 Pavia, Italy
| | - Livia Visai
- Department of Molecular Medicine, Centre for Health Technologies (CHT), INSTM UdR of Pavia, University of Pavia, 27100 Pavia, Italy
- Medicina Clinica-Specialistica, UOR5 Laboratorio di Nanotecnologie, ICS Maugeri, IRCCS, 27100 Pavia, Italy
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Zheng K, Hong W, Ye H, Zhou Z, Ling S, Li Y, Dai Y, Zhong Z, Yang Z, Zheng Y. Chito-oligosaccharides and macrophages have synergistic effects on improving ovarian stem cells function by regulating inflammatory factors. J Ovarian Res 2023; 16:76. [PMID: 37060101 PMCID: PMC10103396 DOI: 10.1186/s13048-023-01143-z] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2022] [Accepted: 03/19/2023] [Indexed: 04/16/2023] Open
Abstract
BACKGROUND Chronic low-grade inflammation and ovarian germline stem cells (OGSCs) aging are important reasons for the decline of ovarian reserve function, resulting in ovarian aging and infertility. Regulation of chronic inflammation is expected to promote the proliferation and differentiation of OGSCs, which will become a key means for maintaining and remodeling ovarian function. Our previous study demonstrated that Chitosan Oligosaccharides (Cos) promoted the OGSCs proliferation and remodelled the ovarian function through improving the secretion of immune related factors,but the mechanism remains unclear, and the role of macrophages, the important source of various inflammatory mediators in the ovary needs to be further studied. In this study, we used the method of macrophages and OGSCs co-culture to observe the effect and mechanism of Cos on OGSCs, and explore what contribution macrophages give during this process. Our finding provides new drug treatment options and methods for the prevention and treatment of premature ovarian failure and infertility. METHODS We used the method of macrophages and OGSCs co-culture to observe the effect and mechanism of Cos on OGSCs, and explore the important contribution of macrophages in it. The immunohistochemical staining was used to locate the OGSCs in the mouse ovary. Immunofluorescent staining, RT-qPCR and ALP staining were used to identify the OGSCs. CCK-8 and western blot were used to evaluate the OGSCs proliferation. β-galactosidase(SA-β-Gal) staining and western blot were used to detect the changing of cyclin-dependent kinase inhibitor 1A(P21), P53, Recombinant Sirtuin 1(SIRT1) and Recombinant Sirtuin 3(SIRT3). The levels of immune factors IL-2, IL-10, TNF-α and TGF-β were explored by using Western blot and ELISA. RESULTS We found that Cos promoted OGSCs proliferation in a dose-and time-dependent manner, accompanied by IL-2, TNF-α increase and IL-10, TGF-β decrease. Mouse monocyte-macrophages Leukemia cells(RAW) can also produce the same effect as Cos. When combined with Cos, it can enhance the proliferative effect of Cos in OGSCs, and further increase IL-2, TNF-α and further decrease IL-10, TGF-β. The macrophages can enhance the proliferative effect of Cos in OGSCs is also associated with the further increase in IL-2, TNF-α and the further decrease in IL-10, TGF-β. In this study, we determined that the anti-aging genes SIRT-1 and SIRT-3 protein levels were increased by Cos and RAW respectively, whereas the senescence-associated SA-β-Gal and aging genes P21 and P53 were decreased. Cos and RAW had a protective effect on OGSCs delaying aging. Furthermore, RAW can further decrease the SA-β-Gal and aging genes P21 and P53 by Cos, and further increase SIRT1 and SIRT3 protein levels in OGSCs by Cos. CONCLUSION In conclusion, Cos and macrophages have synergistic effects on improving OGSCs function and delaying ovarian aging by regulating inflammatory factors.
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Affiliation(s)
- K Zheng
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, China
| | - Wenli Hong
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, China
- Shenzhen University Health Science Center, Shenzhen, China
| | - Haifeng Ye
- Institute of Regenerative Biology and Medicine, Helmholtz Zentrum München, München, Germany
| | - Ziqiong Zhou
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, China
| | - Shuyi Ling
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, China
| | - Yuan Li
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, China
| | - Yuqing Dai
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, China
| | - Zhisheng Zhong
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, China
| | - Ziwei Yang
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, China.
| | - Yuehui Zheng
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, China.
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ÜNAL MS, SEÇME M. Does the ovarian surface epithelium differentiate into primordial follicle and primary follicle precursor structures? CUKUROVA MEDICAL JOURNAL 2022. [DOI: 10.17826/cumj.1134852] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
Abstract
Purpose: The aim of this study is to investigate the differentiation capacity of ovarian surface epithelial cells both in cell culture conditions and in ovarian tissue sections.
Materials and Methods: The ovaries of two prepubertal (4 weeks old) female rats were divided into small pieces and explant cell culture was created. Ovarian surface epithelium proliferating together with ovarian stromal cells in mixed cell culture was isolated and reproduced. In addition, ovarian surface epithelium was examined in histological sections of ovarian tissue and images were taken under the microscope.
Results: The morphological appearance of the ovarian surface epithelium was found to be cobblestone. In the count performed under phase contrast microscopy, it was observed that 2x106 and 3x106 cells were grown in the culture dishes, respectively. Primordial follicle-like structures were observed in some areas of the petri dishes. On the histological sections, primordial and primary follicle precursor structures were observed on the basement membrane.
Conclusion: Showing oocyte markers (Gdf-9, C-Mos, Zpc, Stella) and germ cell markers (Dazl, Vasa, Blimp1, Fragilis) both in cell cultures and in histological sections can give us valuable information in terms of monitoring the differentiation capacity of these cells.
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Hong W, Wang B, Zhu Y, Wu J, Qiu L, Ling S, Zhou Z, Dai Y, Zhong Z, Zheng Y. Female germline stem cells: aging and anti-aging. J Ovarian Res 2022; 15:79. [PMID: 35787298 PMCID: PMC9251950 DOI: 10.1186/s13048-022-01011-2] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2022] [Accepted: 06/17/2022] [Indexed: 01/17/2023] Open
Abstract
The delay of ovarian aging and the fertility preservation of cancer patients are the eternal themes in the field of reproductive medicine. Acting as the pacemaker of female physiological aging, ovary is also considered as the principle player of cancer, cardiovascular diseases, cerebrovascular diseases, neurodegenerative diseases and etc. However, its aging mechanism and preventive measures are still unclear. Some researchers attempt to activate endogenous ovarian female germline stem cells (FGSCs) to restore ovarian function, as the most promising approach. FGSCs are stem cells in the adult ovaries that can be infinitely self-renewing and have the potential of committed differention. This review aims to elucidate FGSCs aging mechanism from multiple perspectives such as niches, immune disorder, chronic inflammation and oxidative stress. Therefore, the rebuilding nichs of FGSCs, regulation of immune dysfunction, anti-inflammation and oxidative stress remission are expected to restore or replenish FGSCs, ultimately to delay ovarian aging.
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Affiliation(s)
- Wenli Hong
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, Guangdong, 518000, People's Republic of China.,Shenzhen University Health Science Center, Shenzhen, Guangdong, 518000, People's Republic of China
| | - Baofeng Wang
- ARTcenter, Shenzhen Hengsheng Hospital, Shenzhen, Guangdong, 518000, People's Republic of China
| | - Yasha Zhu
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, Guangdong, 518000, People's Republic of China
| | - Jun'e Wu
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, Guangdong, 518000, People's Republic of China
| | - Li Qiu
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, Guangdong, 518000, People's Republic of China
| | - Shuyi Ling
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, Guangdong, 518000, People's Republic of China
| | - Ziqiong Zhou
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, Guangdong, 518000, People's Republic of China
| | - Yuqing Dai
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, Guangdong, 518000, People's Republic of China
| | - Zhisheng Zhong
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, Guangdong, 518000, People's Republic of China.
| | - Yuehui Zheng
- Reproductive Health Department, Shenzhen Traditional Chinese Medicine Hospital, the Fourth Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Shenzhen, Guangdong, 518000, People's Republic of China.
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Athar F, Templeman NM. C. elegans as a model organism to study female reproductive health. Comp Biochem Physiol A Mol Integr Physiol 2022; 266:111152. [PMID: 35032657 DOI: 10.1016/j.cbpa.2022.111152] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2021] [Revised: 01/04/2022] [Accepted: 01/07/2022] [Indexed: 12/17/2022]
Abstract
Female reproductive health has been historically understudied and underfunded. Here, we present the advantages of using a free-living nematode, Caenorhabditis elegans, as an animal system to study fundamental aspects of female reproductive health. C. elegans is a powerful high-throughput model organism that shares key genetic and physiological similarities with humans. In this review, we highlight areas of pressing medical and biological importance in the 21st century within the context of female reproductive health. These include the decline in female reproductive capacity with increasing chronological age, reproductive dysfunction arising from toxic environmental insults, and cancers of the reproductive system. C. elegans has been instrumental in uncovering mechanistic insights underlying these processes, and has been valuable for developing and testing therapeutics to combat them. Adopting a convenient model organism such as C. elegans for studying reproductive health will encourage further research into this field, and broaden opportunities for making advancements into evolutionarily conserved mechanisms that control reproductive function.
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Affiliation(s)
- Faria Athar
- Department of Biology, University of Victoria, Victoria, British Columbia V8P 5C2, Canada
| | - Nicole M Templeman
- Department of Biology, University of Victoria, Victoria, British Columbia V8P 5C2, Canada.
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Moghadam ARE, Moghadam MT, Hemadi M, Saki G. Oocyte quality and aging. JBRA Assist Reprod 2022; 26:105-122. [PMID: 34338482 PMCID: PMC8769179 DOI: 10.5935/1518-0557.20210026] [Citation(s) in RCA: 48] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2020] [Accepted: 04/15/2021] [Indexed: 11/20/2022] Open
Abstract
It is well known that female reproduction ability decreases during the forth decade of life due to age-related changes in oocyte quality and quantity; although the number of women trying to conceive has today increased remarkably between the ages of 36 to 44. The causes of reproductive aging and physiological aspects of this phenomenon are still elusive. With increase in the women's age, during Assisted Reproductive Technologies (ART) we have perceived a significant decline in the number and quality of retrieved oocytes, as well as in ovarian follicle reserves. This is because of increased aneuploidy due to factors such as spindle apparatus disruption; oxidative stress and mitochondrial damage. The aim of this review paper is to study data on the potential role of the aging process impacting oocyte quality and female reproductive ability. We present the current evidence that show the decreased oocyte quality with age, related to reductions in female reproductive outcome. The aging process is complicated and it is caused by many factors that control cellular and organism life span. Although the factors responsible for reduced oocyte quality remain unknown, the present review focuses on the potential role of ovarian follicle environment, oocyte structure and its organelles. To find a way to optimize oocyte quality and ameliorate clinical outcomes for women with aging-related causes of infertility.
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Affiliation(s)
- Ali Reza Eftekhari Moghadam
- Cellular & Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
- Department of Anatomical Science, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Mahin Taheri Moghadam
- Department of Anatomical Science, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Masoud Hemadi
- Department of Anatomical Science, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Ghasem Saki
- Department of Anatomical Science, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
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Sequeira RC, Sittadjody S, Criswell T, Atala A, Jackson JD, Yoo JJ. Enhanced method to select human oogonial stem cells for fertility research. Cell Tissue Res 2021; 386:145-156. [PMID: 34415395 DOI: 10.1007/s00441-021-03464-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2019] [Accepted: 04/13/2021] [Indexed: 11/25/2022]
Abstract
Alternative methods to obtain mature oocytes are still needed for women with premature ovarian failure (POF). Oogonial stem cells (OSCs), found in adult ovaries, have provided insight into potential paths to treating infertility. Previously, the DDX4 antibody marker alone was utilized to isolate OSCs; however, extensive debate over its location in OSCs versus resulting oocytes (transmembrane or intracytoplasmic) has raised doubt about the identity of these cells. Separate groups, however, have efficiently isolated OSCs using another antibody marker Ifitm3 which is consistently recognized to be transmembrane in location. We hypothesized that by using anti-DDX4 and anti-IFITM3 antibodies, in combination, with MACS, we would improve the yield of isolated OSCs versus using anti-DDX4 antibodies alone. Our study supports earlier findings of OSCs in ovaries during the entire female lifespan: from reproductive age through post-menopausal age. MACS sorting ovarian cells using a the two-marker combination yielded a ~ twofold higher percentage of OSCs from a given mass of ovarian tissue compared to existing single marker methods while minimizing the debate surrounding germline marker selection. During in vitro culture, isolated cells retained the germline phenotype expression of DDX4 and IFITM3 as confirmed by gene expression analysis, demonstrated characteristic germline stem cell self-assembly into embryoid bodies, and formed > 40 µm "oocyte-like" structures that expressed the early oocyte markers GDF9, DAZL, and ZP1. This enhanced and novel method is clinically significant as it could be utilized in the future to more efficiently produce mature, secondary oocytes, for use with IVF/ICSI to treat POF patients.
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Affiliation(s)
- Russel C Sequeira
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, 27101, USA.
| | - Sivanandane Sittadjody
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, 27101, USA
| | - Tracy Criswell
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, 27101, USA
| | - Anthony Atala
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, 27101, USA
| | - John D Jackson
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, 27101, USA
| | - James J Yoo
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, 27101, USA
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Stucker S, De Angelis J, Kusumbe AP. Heterogeneity and Dynamics of Vasculature in the Endocrine System During Aging and Disease. Front Physiol 2021; 12:624928. [PMID: 33767633 PMCID: PMC7987104 DOI: 10.3389/fphys.2021.624928] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2020] [Accepted: 02/09/2021] [Indexed: 12/11/2022] Open
Abstract
The endocrine system consists of several highly vascularized glands that produce and secrete hormones to maintain body homeostasis and regulate a range of bodily functions and processes, including growth, metabolism and development. The dense and highly vascularized capillary network functions as the main transport system for hormones and regulatory factors to enable efficient endocrine function. The specialized capillary types provide the microenvironments to support stem and progenitor cells, by regulating their survival, maintenance and differentiation. Moreover, the vasculature interacts with endocrine cells supporting their endocrine function. However, the structure and niche function of vasculature in endocrine tissues remain poorly understood. Aging and endocrine disorders are associated with vascular perturbations. Understanding the cellular and molecular cues driving the disease, and age-related vascular perturbations hold potential to manage or even treat endocrine disorders and comorbidities associated with aging. This review aims to describe the structure and niche functions of the vasculature in various endocrine glands and define the vascular changes in aging and endocrine disorders.
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Affiliation(s)
| | | | - Anjali P. Kusumbe
- Tissue and Tumor Microenvironments Group, Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences (NDORMS), University of Oxford, Oxford, United Kingdom
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Huang Y, Ye H, Zhu F, Hu C, Zheng Y. The role of Chito-oligosaccharide in regulating ovarian germ stem cells function and restoring ovarian function in chemotherapy mice. Reprod Biol Endocrinol 2021; 19:14. [PMID: 33494759 PMCID: PMC7830852 DOI: 10.1186/s12958-021-00699-z] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/08/2020] [Accepted: 01/08/2021] [Indexed: 12/31/2022] Open
Abstract
In recent years, the discovery of ovarian germ stem cells (OGSCs) has provided a new research direction for the treatment of female infertility. The ovarian microenvironment affects the proliferation and differentiation of OGSCs, and immune cells and related cytokines are important components of the microenvironment. However, whether improving the ovarian microenvironment can regulate the proliferation of OGSCs and remodel ovarian function has not been reported. In this study, we chelated chito-oligosaccharide (COS) with fluorescein isothiocyanate (FITC) to track the distribution of COS in the body. COS was given to mice through the best route of administration, and the changes in ovarian and immune function were detected using assays of organ index, follicle counting, serum estrogen (E2) and anti-Mullerian hormone (AMH) levels, and the expression of IL-2 and TNF-α in the ovaries. We found that COS significantly increased the organ index of the ovary and immune organs, reduced the rate of follicular atresia, increased the levels of E2 and AMH hormones, and increased the protein expression of IL-2 and TNF-α in the ovary. Then, COS and OGSCs were co-cultured to observe the combination of COS and OGSCs, and measure the survival rate of OGSCs. With increasing time, the fluorescence intensity of cells gradually increased, and the cytokines IL-2 and TNF-α significantly promoted the proliferation of OGSCs. In conclusion, COS could significantly improve the ovarian and immune function of chemotherapy model mice, and improve the survival rate of OGSCs, which provided a preliminary blueprint for further exploring the mechanism of COS in protecting ovarian function.
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Affiliation(s)
- Yaoqi Huang
- Department of Obstetrics & Gynecology, the Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China
| | - Haifeng Ye
- Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum München, Munich, Germany
| | - Feiyin Zhu
- Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, China
| | - Chuan Hu
- Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi, China.
| | - Yuehui Zheng
- Department of reproductive health, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, Guangdong, China.
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Taheri M, Saki G, Nikbakht R, Eftekhari AR. Bone morphogenetic protein 15 induces differentiation of mesenchymal stem cells derived from human follicular fluid to oocyte-like cell. Cell Biol Int 2020; 45:127-139. [PMID: 32997425 DOI: 10.1002/cbin.11475] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2020] [Revised: 09/27/2020] [Accepted: 09/28/2020] [Indexed: 12/12/2022]
Abstract
Follicular fluid (FF) is essential for developing ovarian follicles. Besides the oocytes, FF has abundant undifferentiated somatic cells containing stem cell properties, which are discarded in daily medical procedures. Earlier studies have shown that FF cells could differentiate into primordial germ cells via forming embryoid bodies, which produced oocyte-like cells (OLC). This study aimed at isolating mesenchymal stem cells (MSC) from FF and evaluating the impacts of bone morphogenetic protein 15 (BMP15) on the differentiation of these cells into OLCs. Human FF-derived cells were collected from 78 women in the assisted fertilization program and cultured in human recombinant BMP15 medium for 21 days. Real-time polymerase chain reaction and immunocytochemistry staining characterized MSCs and OLCs. MSCs expressed germline stem cell (GSC) markers, such as OCT4 and Nanog. In the control group, after 15 days, OLCs were formed and expressed zona pellucida markers (ZP2 and ZP3), and reached 20-30 µm in diameter. Ten days after induction with BMP15, round cells developed, and the size of OLCs reached 115 µm. A decrease ranged from 0.04 to 4.5 in the expression of pluripotency and oocyte-specific markers observed in the cells cultured in a BMP15-supplemented medium. FF-derived MSCs have an innate potency to differentiate into OLCs, and BMP15 is effective in promoting the differentiation of these cells, which may give an in vitro model to examine germ cell development.
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Affiliation(s)
- Mahin Taheri
- Cellular and Molecular Research Center, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.,Department of Anatomical Science, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.,Fertility, Infertility and Perinatology Center, Imam Khomeini Hospital, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Ghasem Saki
- Department of Anatomical Science, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Roshan Nikbakht
- Fertility, Infertility and Perinatology Center, Imam Khomeini Hospital, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Ali R Eftekhari
- Cellular and Molecular Research Center, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.,Department of Anatomical Science, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
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12
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Soleimani A, Fard NZ, Talaei-Khozani T, Bahmanpour S. Epidermal growth factor and three-dimensional scaffolds provide conducive environment for differentiation of mouse embryonic stem cells into oocyte-like cells. Cell Biol Int 2020; 44:1850-1859. [PMID: 32437076 DOI: 10.1002/cbin.11391] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2019] [Revised: 04/07/2020] [Accepted: 05/18/2020] [Indexed: 11/09/2022]
Abstract
Three-dimensional (3D) culture provides a biomimicry of the naive microenvironment that can support cell proliferation, differentiation, and regeneration. Some growth factors, such as epidermal growth factor (EGF), facilitate normal meiosis during oocyte maturation in vivo. In this study, a scaffold-based 3D coculture system using purified alginate was applied to induce oocyte differentiation from mouse embryonic stem cells (mESCs). mESCs were induced to differentiate into oocyte-like cells using embryoid body protocol in the two-dimensional or 3D microenvironment in vitro. To increase the efficiency of the oocyte-like cell differentiation from mESCs, we employed a coculture system using ovarian granulosa cells in the presence or absence of epidermal growth factor (+EGF or -EGF) for 14 days and then the cells were assessed for germ cell differentiation, meiotic progression, and oocyte maturation markers. The cultures exposed to EGF in the alginate-based 3D microenvironment showed the highest level of premeiotic (Oct4 and Mvh), meiotic (Scp1, Scp3, Stra8, and Rec8), and oocyte maturation (Gdf9, Cx37, and Zp2) marker genes (p < .05) in comparison to other groups. According to the gene-expression patterns, we can conclude that alginate-based 3D coculture system provided a highly efficient protocol for oocyte-like cell differentiation from mESCs. The data showed that this culture system along with EGF improved the rate of in vitro oocyte-like cell differentiation.
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Affiliation(s)
- Azam Soleimani
- Stem Cell Research Laboratory, Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Nehleh Zarei Fard
- Stem Cell Research Laboratory, Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Tahereh Talaei-Khozani
- Stem Cell Research Laboratory, Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Soghra Bahmanpour
- Stem Cell Research Laboratory, Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
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13
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Wang Z, Yang Y, Li S, Li K, Tang Z. Analysis and comparison of long non‐codingRNAs expressed in the ovaries of Meishan and Yorkshire pigs. Anim Genet 2019; 50:660-669. [DOI: 10.1111/age.12849] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/18/2019] [Indexed: 11/28/2022]
Affiliation(s)
- Z. Wang
- Genome Analysis Laboratory of the Ministry of Agriculture Agricultural Genome Institute at Shenzhen Chinese Academy of Agricultural Sciences Shenzhen 518124 China
- Department of Computer Science City University of Hong Kong Kowloon 999077 Hong Kong
| | - Y. Yang
- Genome Analysis Laboratory of the Ministry of Agriculture Agricultural Genome Institute at Shenzhen Chinese Academy of Agricultural Sciences Shenzhen 518124 China
- Research Centre for Nutriomics State Key Laboratory of Animal Nutrition Institute of Animal Sciences Chinese Academy of Agricultural Sciences Shenzhen 10093 China
| | - S. Li
- Department of Computer Science City University of Hong Kong Kowloon 999077 Hong Kong
| | - K. Li
- Research Centre for Nutriomics State Key Laboratory of Animal Nutrition Institute of Animal Sciences Chinese Academy of Agricultural Sciences Shenzhen 10093 China
| | - Z. Tang
- Genome Analysis Laboratory of the Ministry of Agriculture Agricultural Genome Institute at Shenzhen Chinese Academy of Agricultural Sciences Shenzhen 518124 China
- Research Centre for Nutriomics State Key Laboratory of Animal Nutrition Institute of Animal Sciences Chinese Academy of Agricultural Sciences Shenzhen 10093 China
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14
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Effects of VEGF
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Mesenchymal Stem Cells and Platelet-Rich Plasma on Inbred Rat Ovarian Functions in Cyclophosphamide-Induced Premature Ovarian Insufficiency Model. Stem Cell Rev Rep 2019; 15:558-573. [DOI: 10.1007/s12015-019-09892-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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15
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Besikcioglu HE, Sarıbas GS, Ozogul C, Tiryaki M, Kilic S, Pınarlı FA, Gulbahar O. Determination of the effects of bone marrow derived mesenchymal stem cells and ovarian stromal stem cells on follicular maturation in cyclophosphamide induced ovarian failure in rats. Taiwan J Obstet Gynecol 2019; 58:53-59. [PMID: 30638481 DOI: 10.1016/j.tjog.2018.11.010] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/04/2018] [Indexed: 01/02/2023] Open
Abstract
OBJECTIVE Chemotherapy causes depletion of primordial follicles that leads to premature ovarian failure in female cancer survivals. We investigated the effect of bone marrow derived mesenchymal (BMMSCs) and ovarian stromal stem cells (OSSCs) on follicle maturation in chemotherapy induced ovarian failure. MATERIAL AND METHODS Thirty six Wistar Albino female rats were divided into three groups. Cyclophosphamide at a dose of 200 mg/kg was intraperitoneally (IP) given to the rats in all groups two times. 4 × 106 BMMSCs (IP) was injected to the group-2 and 4 × 106 OSSCs (IP) was injected to the group-3. Serum Anti-Müllerian Hormone (AMH) levels was determined with ELISA and primordial follicles were counted for investigation of primordial follicle reserve. The ovarian structure were evaluated histomorphologically. Localization of BrdU labeled stem cells, the expression of the cell cycle regulator p34Cdc2, gap junction protein p-connexin43 and intraovarian regulators of folliculogenesis Bone Morphogenic Protein 6 and 15 (BMP-6 and BMP-15) were investigated by immunohistochemistry. RESULTS The immunstaining of BMP-6 was higher in oocytes of group-3 more than group-1 and group-2. The immunpositivity of p34cdc2 and BMP-15 were also higher in follicular cells of group-3 than the other groups. The presence of p-connexin43 in group-3 was determined more than group-1 and group-2. The ovarian follicles with normal histological structure were observed just in group-3. Although, The AMH levels were decreased in rats from all groups at the end of experimental procedure the primordial follicle counts in group-3 was significantly higher than group-1. CONCLUSION Our findings suggest that OSSCs have more protective effect on follicle maturation than BMMSCs in cyclophosphamide induced ovarian damage.
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Affiliation(s)
| | - Gulistan Sanem Sarıbas
- Department of Histology and Embryology, Faculty of Medicine, Kırşehir Ahi Evran University, Kırşehir, Turkey
| | - Candan Ozogul
- Department of Histology and Embryology, Faculty of Medicine Gazi University, Ankara, Turkey.
| | - Meral Tiryaki
- Department of Pathology, Yıldırım Beyazıt Training and Research Hospital, Ankara, Turkey
| | - Sevtap Kilic
- Department of Obstetrics and Gynecology, Faculty of Medicine, Bahçeşehir University, Istanbul, Turkey
| | - Ferda Alpaslan Pınarlı
- Genetic Diagnostic Center, Yıldırım Beyazıt Training and Research Hospital, Ankara, Turkey
| | - Ozlem Gulbahar
- Department of Medical Biochemistry, Faculty of Medicine, Gazi University, Ankara, Turkey
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16
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Ilyina AP, Krasnov MS, Elistratov PA, Yamskova NV, Yamskova VP, Buryak AK, Yamskov IA. Study of the Physicochemical and Biological Properties of a Protein–Peptide Complex Isolated from Cattle Ovarian Tissue. APPL BIOCHEM MICRO+ 2019. [DOI: 10.1134/s000368381901006x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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17
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Liu Y, Xu J, Zhu F, Ye H, Hu C, Huang J, Zheng Y. Research advances in the regulation of the putative ovarian germline stem cell niche on female germline stem cells. Syst Biol Reprod Med 2018; 65:121-128. [PMID: 30204491 DOI: 10.1080/19396368.2018.1515272] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Stem cells are ideal seeding cells, which have the potential for self-renewal and multiple differentiation, and they play a fundamental role in maintaining homeostasis and regenerating and repairing tissue. The discovery of female germline stem cells (FGSCs) brings much hope for the postnatal renewal of oocytes and solving some female infertility problems. Ovarian function declines with increasing female age. Moreover, ovarian germline stem cell niche-aging could be the main cause of ovarian senescence, which ultimately leads to decreased follicle generation, declining female fertility, and age-related diseases, such as osteoporosis and ovarian cancer. The ovarian germline stem cell niche is the surrounding microenvironment in which FGSCs live, and it helps control the biological characteristics of FGSCs in many ways, such as nutritional supply and immunological cytokine secretion. This paper reviews the knowledge about the ovarian germline stem cell niche and its probable regulatory mechanisms on FGSCs, which provides valuable scientific information and scope for the prevention and treatment of ovarian senescence. Abbreviations: BMP: bone morphogenetic protein; Dpp: decapentaplegic; FGSC: female germline stem cell; IL, interleukin; OGSC: ovarian germline stem cells; ROS: reactive oxygen species; TGF, transforming growth factor; TNF, tumor necrosis factor.
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Affiliation(s)
- Yangchun Liu
- a Jiangxi Medical College , Nanchang University , Nanchang , Jiangxi , PR China.,b Queen Mary College of Nanchang University , Nanchang , Jiangxi , PR China
| | - Jiao Xu
- a Jiangxi Medical College , Nanchang University , Nanchang , Jiangxi , PR China.,c First Clinical College of Nanchang University , Nanchang , Jiangxi , PR China
| | - Feiyin Zhu
- a Jiangxi Medical College , Nanchang University , Nanchang , Jiangxi , PR China.,b Queen Mary College of Nanchang University , Nanchang , Jiangxi , PR China
| | - Haifeng Ye
- a Jiangxi Medical College , Nanchang University , Nanchang , Jiangxi , PR China.,d The Key Laboratory of Reproductive Physiology and Pathology of Jiangxi Province , Nanchang , Jiangxi , PR China
| | - Chuan Hu
- a Jiangxi Medical College , Nanchang University , Nanchang , Jiangxi , PR China.,d The Key Laboratory of Reproductive Physiology and Pathology of Jiangxi Province , Nanchang , Jiangxi , PR China
| | - Jian Huang
- a Jiangxi Medical College , Nanchang University , Nanchang , Jiangxi , PR China.,d The Key Laboratory of Reproductive Physiology and Pathology of Jiangxi Province , Nanchang , Jiangxi , PR China
| | - Yuehui Zheng
- a Jiangxi Medical College , Nanchang University , Nanchang , Jiangxi , PR China.,d The Key Laboratory of Reproductive Physiology and Pathology of Jiangxi Province , Nanchang , Jiangxi , PR China
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18
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Sills ES, Rickers NS, Li X, Palermo GD. First data on in vitro fertilization and blastocyst formation after intraovarian injection of calcium gluconate-activated autologous platelet rich plasma. Gynecol Endocrinol 2018; 34:756-760. [PMID: 29486615 DOI: 10.1080/09513590.2018.1445219] [Citation(s) in RCA: 76] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/17/2022] Open
Abstract
Platelets modulate clinically relevant yet incompletely understood tissue regeneration processes, and platelet rich plasma (PRP) has been previously used with some success in various non-reproductive medical contexts. Here, we extended PRP application to ovarian tissue with a view to document impact on ovarian reserve among women attending for infertility treatment. PRP was freshly isolated from patients (n= 4) with diminished ovarian reserve as determined by at least one prior IVF cycle canceled for poor follicular recruitment response or estimated by serum AMH and/or FSH, no menses for ≥1 year. Immediately following substrate isolation and activation with calcium gluconate, approximately 5 mL of autologous PRP was injected into each ovary under direct transvaginal sonogram guidance. For each study subject, AMH, FSH, and serum estradiol data were recorded at two-week intervals post-PRP and compared to baseline (pre-PRP) values. In this pilot group, mean (±SD) patient age was 42 ± 4 years with infertility duration reported as 60 ± 25 months. Following this protocol of intraovarian PRP administration, increases in serum AMH (p = .17), decreases in FSH (p < .01), or both, were observed in all cases, sufficient to permit retrieval of 5.3 ± 1.3 MII oocytes. IVF occurred 78 ± 22 (range = 59-110) days after activated PRP injection, and results appeared independent of patient age, infertility duration, baseline platelet concentration or pretreatment antral follicle count. Each patient had at least one blastocyst suitable for cryopreservation. While autologous PRP has been successfully applied therapeutically to various tissues to accelerate healing and wound repair, this is the first description of direct injection of activated PRP into the human ovary of poor prognosis IVF patients. Evidence of improved ovarian function was noted in all who received intraovarian PRP, possibly as early as two months after treatment. Additional research is needed to clarify (and enhance) which PRP components are responsible for altered ovarian function, and to identify predictive characteristics for patients most likely to benefit from this intervention.
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Affiliation(s)
- E Scott Sills
- a Office for Reproductive Research , Center for Advanced Genetics , Carlsbad , CA , USA
- b Applied Biotechnology Research Group , University of Westminster , London, UK
| | - Natalie S Rickers
- a Office for Reproductive Research , Center for Advanced Genetics , Carlsbad , CA , USA
| | - Xiang Li
- a Office for Reproductive Research , Center for Advanced Genetics , Carlsbad , CA , USA
- c Data Analytics, Paralian Technology, Inc , Mission Viejo , CA , USA
| | - Gianpiero D Palermo
- d Center for Reproductive Medicine & Infertility, Weill Medical College of Cornell University , New York , NY , USA
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19
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Characterization of female germline stem cells from adult mouse ovaries and the role of rapamycin on them. Cytotechnology 2018; 70:843-854. [PMID: 29372468 DOI: 10.1007/s10616-018-0196-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2017] [Accepted: 01/17/2018] [Indexed: 12/13/2022] Open
Abstract
Germline stem cells (GSCs) play an indispensable role in establishing the fertility of an organism. The isolation and culture of adult female GSCs (FGSCs) have provided a robust foundation to study the development of female germ cells in rodents. However, many problems still need to be identified, such as the origin and location of FGSCs and the specific markers for screening. In this study, we acquired FGSCs that stably expressed Oct4 from Oct4 promoter-GFP transgenic mouse ovarian surface epithelium and cortical layer, and identified the cells possessing the representative features including the expression of GSCs marker genes and the potentiality of differentiation into all three germ layers in vitro. Moreover, rapamycin was confirmed to promote proliferation of mouse FGSCs and inhibit the differentiation capability in vivo. In addition to the reported disinfection function, rapamycin inhibited the activation of primordial follicles, as the inhibitor of mechanistic target of rapamycin pathway. These results will contribute to the study on folliculogenesis or oogenesis mechanism and have important implications on developing new technology and therapeutic approach in medicine for premature ovarian failure, infertility and even ovary remodelling in future.
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20
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Yazdekhasti H, Hosseini MA, Rajabi Z, Parvari S, Salehnia M, Koruji M, Izadyar F, Aliakbari F, Abbasi M. Improved Isolation, Proliferation, and Differentiation Capacity of Mouse Ovarian Putative Stem Cells. Cell Reprogram 2017; 19:132-144. [PMID: 28375748 DOI: 10.1089/cell.2016.0054] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
The recent discovery of ovarian stem cells in postnatal mammalian ovaries, also referred to as putative stem cells (PSCs), and their roles in mammalian fertility has challenged the long-existing theory that women are endowed with a certain number of germ cells. The rare amount of PSCs is the major limitation for utilizing them through different applications. Therefore, this study was conducted in six phases to find a way to increase the number of Fragilis- and mouse vasa homolog (MVH)-positive sorted cells from 14-day-old NMRI strain mice. Results showed that there is a population of Fragilis- and MVH-positive cells with pluripotent stem cell characteristics, which can be isolated and expanded for months in vitro. PSCs increase their proliferation capacity under the influence of some mitogenic agents, and our results showed that different doses of stem cell factor (SCF) induce PSC proliferation with the maximum increase observed at 50 ng/mL. SCF was also able to increase the number of Fragilis- and MVH-positive cells after sorting by magnetic-activated cell sorting and enhance colony formation efficiency in sorted cells. Differentiation capacity assay indicated that there is a basic level of spontaneous differentiation toward oocyte-like cells during 3 days of culture. However, relative gene expression was significantly higher in the follicle-stimulating hormone-treated groups, especially in the Fragilis- sorted PSCs. We suggest that higher number of PSCs provides us either a greater source of energy that can be injected into energy-impaired oocytes in women with a history of repeat IVF failure or a good source for research.
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Affiliation(s)
- Hossein Yazdekhasti
- 1 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences , Tehran, Iran
| | - Marzieh Agha Hosseini
- 2 Department of Infertility, Shariati Hospital, Tehran University of Medical Sciences , Tehran, Iran
| | - Zahra Rajabi
- 1 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences , Tehran, Iran
| | - Soraya Parvari
- 3 Department of Anatomy, School of Medicine, Alborz University of Medical Sciences , Karaj, Iran
| | - Mojdeh Salehnia
- 4 Department of Anatomy, School of Medical Sciences, Tarbiat Modarres University , Tehran, Iran
| | - Morteza Koruji
- 5 Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences , Tehran, Iran
| | | | - Fereshte Aliakbari
- 1 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences , Tehran, Iran
| | - Mehdi Abbasi
- 1 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences , Tehran, Iran
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21
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Abstract
Endometriosis is defined by the presence of endometrial ectopia. Multiple hypotheses have been postulated to explain the etiology of endometriosis to understand various clinical evidences. The etiology of endometriosis is still unclear.The primary question to understanding the etiology of endometrial ectopia (endometriosis) is determining the origin of eutopic (normally cited) endometrium.According to the new theory, primordial germ cells migrate from hypoblast (yolk sac close to the allantois) to the gonadal ridges. The gonadal ridges which composed of primordial germ cells derive to the: eutopic endometrium, ovary, ovarian ligament and ligamentum teres uteri.There are 2 principal processes in uterine organogenesis: the intersection of gonadal ridges with mesonephral ducts to form the uterine folds with an endometrial cavity and the fusion of the both uterine folds together to form the unicavital (normal) uterus. In the uterine folds there are closer cell-to-cell communications, polypotential germ cells differentiate and grow into myometrium and endometrial layers.Some of the polypotential germ cells fail to reach the ridges and stay in the peritoneal cavity, where they may be transforming into external endometrial heterotopies.The main insight in the etiology of endometriosis is polypotential germ cells origin, which may explain its potency, pathogenesis and expansion.
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Affiliation(s)
- Zograb Makiyan
- a Department of Operative Gynaecology , Federal State Scientific Centre of Obstetrics, Gynaecology and Perinatology after V.I. Kulakov , Moscow , Russia
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22
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Bukovsky A. Novel Immunological Aspects for the Treatment of Age-induced Ovarian and Testicular Infertility, Other Functional Diseases, and Early and Advanced Cancer Immunotherapy. Hum Reprod 2016. [DOI: 10.1002/9781118849613.ch4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Affiliation(s)
- Antonin Bukovsky
- The Laboratory of Reproductive Biology BIOCEV, Institute of Biotechnology; Academy of Sciences of the Czech Republic; Prague Czech Republic
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23
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Bukovsky A. Involvement of blood mononuclear cells in the infertility, age-associated diseases and cancer treatment. World J Stem Cells 2016; 8:399-427. [PMID: 28074124 PMCID: PMC5183987 DOI: 10.4252/wjsc.v8.i12.399] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/12/2016] [Revised: 08/19/2016] [Accepted: 09/21/2016] [Indexed: 02/06/2023] Open
Abstract
Blood mononuclear cells consist of T cells and monocyte derived cells. Beside immunity, the blood mononuclear cells belong to the complex tissue control system (TCS), where they exhibit morphostatic function by stimulating proliferation of tissue stem cells followed by cellular differentiation, that is stopped after attaining the proper functional stage, which differs among various tissue types. Therefore, the term immune and morphostatic system (IMS) should be implied. The TCS-mediated morphostasis also consists of vascular pericytes controlled by autonomic innervation, which is regulating the quantity of distinct tissues in vivo. Lack of proper differentiation of tissue cells by TCS causes either tissue underdevelopment, e.g., muscular dystrophy, or degenerative functional failures, e.g., type 1 diabetes and age-associated diseases. With the gradual IMS regression after 35 years of age the gonadal infertility develops, followed by a growing incidence of age-associated diseases and cancers. Without restoring an altered TCS function in a degenerative disease, the implantation of tissue-specific stem cells alone by regenerative medicine can not be successful. Transfused young blood could temporarily restore fertility to enable parenthood. The young blood could also temporarily alleviate aging diseases, and this can be extended by substances inducing IMS regeneration, like the honey bee propolis. The local and/or systemic use of honey bee propolis stopped hair and teeth loss, regressed varicose veins, improved altered hearing, and lowered high blood pressure and sugar levels. Complete regression of stage IV ovarian cancer with liver metastases after a simple elaborated immunotherapy is also reported.
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Affiliation(s)
- Antonin Bukovsky
- Antonin Bukovsky, Laboratory of Reproductive Biology BIOCEV, Institute of Biotechnology Czech Academy of Sciences, 25250 Vestec, Czech Republic
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24
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Yazdekhasti H, Rajabi Z, Parvari S, Abbasi M. Used protocols for isolation and propagation of ovarian stem cells, different cells with different traits. J Ovarian Res 2016; 9:68. [PMID: 27765047 PMCID: PMC5072317 DOI: 10.1186/s13048-016-0274-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2016] [Accepted: 10/03/2016] [Indexed: 11/10/2022] Open
Abstract
Although existence of ovarian stem cells (OSCs) in mammalian postnatal ovary is still under controversy, however, it has been almost accepted that OSCs are contributing actively to folliculogenesis and neo-oogenesis. Recently, various methods with different efficacies have been employed for OSCs isolation from ovarian tissue, which these methods could be chosen depends on aim of isolation and accessible equipments and materials in lab. Although isolated OSCs from different methods have various traits and characterizations, which might become from their different nature and origin, however these stem cells are promising source for woman infertility treatment or source of energy for women with a history of repeat IVF failure in near future. This review has brought together and summarized currently used protocols for isolation and propagation of OSCs in vitro.
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Affiliation(s)
- Hossein Yazdekhasti
- Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Zahra Rajabi
- Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Soraya Parvari
- Department of Anatomy, Faculty of Medicine, Alborz University of Medical Sciences, Karaj, Iran
| | - Mehdi Abbasi
- Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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25
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Bhartiya D, Shaikh A, Anand S, Patel H, Kapoor S, Sriraman K, Parte S, Unni S. Endogenous, very small embryonic-like stem cells: critical review, therapeutic potential and a look ahead. Hum Reprod Update 2016; 23:41-76. [PMID: 27614362 DOI: 10.1093/humupd/dmw030] [Citation(s) in RCA: 87] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2016] [Revised: 07/27/2016] [Accepted: 08/04/2016] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND Both pluripotent very small embryonic-like stem cells (VSELs) and induced pluripotent stem (iPS) cells were reported in 2006. In 2012, a Nobel Prize was awarded for iPS technology whereas even today the very existence of VSELs is not well accepted. The underlying reason is that VSELs exist in low numbers, remain dormant under homeostatic conditions, are very small in size and do not pellet down at 250-280g. The VSELs maintain life-long tissue homeostasis, serve as a backup pool for adult stem cells and are mobilized under stress conditions. An imbalance in VSELs function (uncontrolled proliferation) may result in cancer. SEARCH METHODS The electronic database 'Medline/Pubmed' was systematically searched with the subject heading term 'very small embryonic-like stem cells'. OBJECTIVE AND RATIONALE The most primitive stem cells that undergo asymmetric cell divisions to self-renew and give rise to progenitors still remain elusive in the hematopoietic system and testes, while the presence of stem cells in ovary is still being debated. We propose to review the available literature on VSELs, the methods of their isolation and characterization, their ontogeny, how they compare with embryonic stem (ES) cells, primordial germ cells (PGCs) and iPS cells, and their role in maintaining tissue homeostasis. The review includes a look ahead on how VSELs will result in paradigm shifts in basic reproductive biology. OUTCOMES Adult tissue-specific stem cells including hematopoietic, spermatogonial, ovarian and mesenchymal stem cells have good proliferation potential and are indeed committed progenitors (with cytoplasmic OCT-4), which arise by asymmetric cell divisions of pluripotent VSELs (with nuclear OCT-4). VSELs are the most primitive stem cells and postulated to be an overlapping population with the PGCs. Rather than migrating only to the gonads, PGCs migrate and survive in various adult body organs throughout life as VSELs. VSELs express both pluripotent and PGC-specific markers and are epigenetically and developmentally more mature compared with ES cells obtained from the inner cell mass of a blastocyst-stage embryo. As a result, VSELs readily differentiate into three embryonic germ layers and spontaneously give rise to both sperm and oocytes in vitro. Like PGCs, VSELs do not divide readily in culture, nor produce teratoma or integrate in the developing embryo. But this property of being relatively quiescent allows endogenous VSELs to survive various kinds of toxic insults. VSELs that survive oncotherapy can be targeted to induce endogenous regeneration of non-functional gonads. Transplanting healthy niche (mesenchymal) cells have resulted in improved gonadal function and live births. WIDER IMPLICATIONS Being quiescent, VSELs possibly do not accumulate genomic (nuclear or mitochondrial) mutations and thus may be ideal endogenous, pluripotent stem cell candidates for regenerative and reproductive medicine. The presence of VSELs in adult gonads and the fact that they survive oncotherapy may obviate the need to bank gonadal tissue for fertility preservation prior to oncotherapy. VSELs and their ability to undergo spermatogenesis/neo-oogenesis in the presence of a healthy niche will help identify newer strategies toward fertility restoration in cancer survivors, delaying menopause and also enabling aged mothers to have better quality eggs.
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Affiliation(s)
- Deepa Bhartiya
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India
| | - Ambreen Shaikh
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India
| | - Sandhya Anand
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India
| | - Hiren Patel
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India
| | - Sona Kapoor
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India
| | - Kalpana Sriraman
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India.,The Foundation for Medical Research, 84-A, RG Thadani Marg, Worli, Mumbai 400018, India
| | - Seema Parte
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India.,Department of Physiology, James Graham Brown Cancer Centre, University of Louisville School of Medicine, 2301 S 3rd St, Louisville, KY 40202, USA
| | - Sreepoorna Unni
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai 400 012, India.,Inter Disciplinary Studies Department, University College, Zayed University, Academic City, PO Box 19282, Dubai, United Arab Emirates
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Ye H, Li X, Zheng T, Liang X, Li J, Huang J, Pan Z, Zheng Y. The effect of the immune system on ovarian function and features of ovarian germline stem cells. SPRINGERPLUS 2016; 5:990. [PMID: 27398269 PMCID: PMC4937004 DOI: 10.1186/s40064-016-2390-3] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/14/2016] [Accepted: 05/23/2016] [Indexed: 01/30/2023]
Abstract
In addition to its role in maintaining organism homeostasis, the immune system also plays a crucial role in the modulation of ovarian function, as it regulates ovarian development, follicular maturation, ovulation and the formation of the corpus luteum. Ovarian germline stem cells are pluripotent stem cells derived from the ovarian cortex that can differentiate into ovarian germ cells and primary granulosa cells. Recent work has demonstrated that the proliferation and differentiation of ovarian germline stem cells is regulated in part by immune cells and their secreted factors. This paper reviews the role of the immune system in the regulation of ovarian function, the relationship between immune components and ovarian germline stem cells and current research efforts in this field.
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Affiliation(s)
- Haifeng Ye
- Medical Teaching Laboratory Center, Jiangxi Medical College, Nanchang University, Nanchang, China ; The Key Laboratory of Reproductive Physiology and Pathology of Jiangxi Province, Nanchang, China
| | - Xiaoyan Li
- Medical Teaching Laboratory Center, Jiangxi Medical College, Nanchang University, Nanchang, China ; The Key Laboratory of Reproductive Physiology and Pathology of Jiangxi Province, Nanchang, China
| | - Tuochen Zheng
- School of the 1st Clinical Medical Sciences, Jiangxi Medical College, Nanchang University, Nanchang, China
| | - Xia Liang
- Medical Teaching Laboratory Center, Jiangxi Medical College, Nanchang University, Nanchang, China ; The Key Laboratory of Reproductive Physiology and Pathology of Jiangxi Province, Nanchang, China
| | - Jia Li
- School of Life Science, Nanchang University, Nanchang, China ; Medical Teaching Laboratory Center, Jiangxi Medical College, Nanchang University, Nanchang, China
| | - Jian Huang
- Medical Teaching Laboratory Center, Jiangxi Medical College, Nanchang University, Nanchang, China ; The Key Laboratory of Reproductive Physiology and Pathology of Jiangxi Province, Nanchang, China
| | - Zezheng Pan
- Medical Teaching Laboratory Center, Jiangxi Medical College, Nanchang University, Nanchang, China ; Faculty of Basic Medical Science, Jiangxi Medical College, Nanchang University, Nanchang, China
| | - Yuehui Zheng
- School of Life Science, Nanchang University, Nanchang, China ; Medical Teaching Laboratory Center, Jiangxi Medical College, Nanchang University, Nanchang, China
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Wear HM, McPike MJ, Watanabe KH. From primordial germ cells to primordial follicles: a review and visual representation of early ovarian development in mice. J Ovarian Res 2016; 9:36. [PMID: 27329176 PMCID: PMC4915180 DOI: 10.1186/s13048-016-0246-7] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2016] [Accepted: 06/13/2016] [Indexed: 01/08/2023] Open
Abstract
Background Normal development of reproductive organs is crucial for successful reproduction. In mice the early ovarian developmental process occurs during the embryonic and postnatal period and is regulated through a series of molecular signaling events. Early ovarian development in mice is a seventeen-day process that begins with the rise of six primordial germ cells on embryonic day five (E5) and ends with the formation of primordial follicles on postnatal day two (P2). Results We reviewed the current literature and created a visual representation of early ovarian development that depicts the important molecular events and associated phenotypic outcomes based on primary data. The visual representation shows the timeline of key signaling interactions and regulation of protein expression in different cells involved in ovarian development. The major developmental events were divided into five phases: 1) origin of germ cells and maintenance of pluripotency; 2) primordial germ cell migration; 3) sex differentiation; 4) formation of germ cell nests; and 5) germ cell nest breakdown and primordial follicle formation. Conclusions This review and visual representation provide a summary of the current scientific understanding of the key regulation and signaling during ovarian development and highlights areas needing further study. The visual representation can be used as an educational resource to link molecular events with phenotypic outcomes; serves as a tool to generate new hypotheses and predictions of adverse reproductive outcomes due to perturbations at the molecular and cellular levels; and provides a comprehendible foundation for computational model development and hypothesis testing.
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Affiliation(s)
- Hannah M Wear
- Institute of Environmental Health, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd. Mail code HRC3, Portland, OR, 97239, USA
| | - Matthew J McPike
- Institute of Environmental Health, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd. Mail code HRC3, Portland, OR, 97239, USA
| | - Karen H Watanabe
- School of Public Health, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd. Mail code GH230, Portland, OR, 97239, USA.
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Elfayomy AK, Almasry SM, El-Tarhouny SA, Eldomiaty MA. Human umbilical cord blood-mesenchymal stem cells transplantation renovates the ovarian surface epithelium in a rat model of premature ovarian failure: Possible direct and indirect effects. Tissue Cell 2016; 48:370-82. [PMID: 27233913 DOI: 10.1016/j.tice.2016.05.001] [Citation(s) in RCA: 69] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2016] [Revised: 05/04/2016] [Accepted: 05/07/2016] [Indexed: 01/09/2023]
Abstract
This study aimed to isolate mesenchymal stem cells (MSC) from human umbilical cord blood (HCB) and to explore their influence on the ovarian epithelium after paclitaxel-induced ovarian failure. Ninety-five rats were divided into 6 groups: control, paclitaxel, paclitaxel and saline, HCB-MSC-treated for 2 weeks, HCB-MSC-treated for 4 weeks, and HCB-MSC-treated for 6 weeks. HCB cells were studied for CD34, CD44, and Oct ¾ using flow cytometry. Serum levels of FSH and E2 were measured using ELISA, RT-PCR analysis for human gene; beta-actin (ACTB), immunohistochemical analysis for CK 8/18, TGF-ß, PCNA and CASP-3 were performed. We found that ACTB gene was expressed in all rats' ovaries received HCB-MSC. After 4 weeks of transplantation, there was significant reduction in FSH, elevation in E2 levels, stabilization of the surface epithelium morphostasis, an increase in the antral follicle count and increase in integrated densities (ID) of CK 8/18, TGF-ß, and PCNA expressions and decrease in ID of CASP-3 expression. We concluded that HCB-MSC can restore the ovarian function after paclitaxel injection through a direct triggering effect on the ovarian epithelium and/or indirect enrichment of ovarian niche through regulating tissue expression of CK 8/18, TGF-ß and PCNA. These molecules are crucial in regulating folliculogenesis and suppressing CASP-3-induced apoptosis.
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Affiliation(s)
- Amr K Elfayomy
- Department of Obstetrics and Gynecology, Taibah University, Almadinah Almunawarah, Saudi Arabia; Department of Obstetrics and Gynecology, Zagazig University, Zagazig, Egypt
| | - Shaima M Almasry
- Department of Anatomy, Taibah University, Almadinah Almunawarah, Saudi Arabia; Department of Anatomy, Mansoura University, Mansoura, Egypt.
| | - Shereen A El-Tarhouny
- Department of Clinical Biochemistry, Taibah University, Saudi Arabia; Department of Medical Biochemistry, Zagazig University, Egypt
| | - Magda A Eldomiaty
- Department of Anatomy, Taibah University, Almadinah Almunawarah, Saudi Arabia; Department of Anatomy, Tanta University, Tanta, Egypt
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Improvement in Isolation and Identification of Mouse Oogonial Stem Cells. Stem Cells Int 2015; 2016:2749461. [PMID: 26635882 PMCID: PMC4655301 DOI: 10.1155/2016/2749461] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2015] [Revised: 06/19/2015] [Accepted: 06/22/2015] [Indexed: 01/16/2023] Open
Abstract
Female germline stem cells (FGSCs) or oogonial stem cells (OSCs) have the capacity to generate newborn oocytes and thus open a new door to fight ovarian aging and female infertility. However, the production and identification of OSCs are difficult for investigators. Rare amount of these cells in the ovary results in the failure of the acquisition of OSCs. Furthermore, the oocyte formation by OSCs in vivo was usually confirmed using tissue sections by immunofluorescence or immunohistochemistry in previous studies. STO or MEF feeder cells are derived from mouse, not human. In our study, we modified the protocol. The cells were digested from ovaries and cultured for 2-3 days and then were purified by magnetic-activated cell sorting (MACS). The ovaries and fetus of mice injected with EGFP-positive OSCs were prepared and put on the slides to directly visualize oocyte and progeny formation under microscope. Additionally, the human umbilical cord mesenchymal stem cells (hUC-MSCs) were also used as feeder cells to support the proliferation of OSCs. The results showed that all the modified procedures can significantly improve and facilitate the generation and characterization of OSCs, and hUC-MSCs as feeder will be useful for isolation and proliferation of human OSCs avoiding contamination from mouse.
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Abstract
In spite of generally accepted dogma that the total number of follicles and oocytes is established in human ovaries during the fetal period of life rather than forming de novo in adult ovaries, some new evidence in the field challenges this understanding. Several studies have shown that different populations of stem cells, such as germinal stem cells and small round stem cells with diameters of 2 to 4 μm, that resembled very small embryonic-like stem cells and expressed several genes related to primordial germ cells, pluripotency, and germinal lineage are present in adult human ovaries and originate in ovarian surface epithelium. These small stem cells were pushed into the germinal direction of development and formed primitive oocyte-like cells in vitro. Moreover, oocyte-like cells were also formed in vitro from embryonic stem cells and induced pluripotent stem cells. This indicates that postnatal oogenesis is not excluded. It is further supported by the occurrence of mesenchymal stem cells that can restore the function of sterilized ovaries and lead to the formation of new follicles and oocytes in animal models. Both oogenesis in vitro and transplantation of stem cell-derived "oocytes" into the ovarian niche to direct their natural maturation represent a big challenge for reproductive biomedicine in the treatment of female infertility in the future and needs to be explored and interpreted with caution, but it is still very important for clinical practice in the field of reproductive medicine.
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Affiliation(s)
- Irma Virant-Klun
- Department of Obstetrics and Gynaecology, University Medical Center Ljubljana, Ljubljana, Slovenia
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31
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Sriraman K, Bhartiya D, Anand S, Bhutda S. Mouse Ovarian Very Small Embryonic-Like Stem Cells Resist Chemotherapy and Retain Ability to Initiate Oocyte-Specific Differentiation. Reprod Sci 2015; 22:884-903. [PMID: 25779995 DOI: 10.1177/1933719115576727] [Citation(s) in RCA: 65] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
This study was undertaken to investigate stem cells in adult mouse ovary, the effect of chemotherapy on them and their potential to differentiate into germ cells. Very small embryonic-like stem cells (VSELs) that were SCA-1+/Lin-/CD45-, positive for nuclear octamer-binding transforming factor 4 (OCT-4), Nanog, and cell surface stage-specific embryonic antigen 1, were identified in adult mouse ovary. Chemotherapy resulted in complete loss of follicular reserve and cytoplasmic OCT-4 positive progenitors (ovarian germ stem cells) but VSELs survived. In ovarian surface epithelial (OSE) cell cultures from chemoablated ovary, proliferating germ cell clusters and mouse vasa homolog/growth differentiation factor 9-positive oocyte-like structure were observed by day 6, probably arising as a result of differentiation of the surviving VSELs. Follicle-stimulating hormone (FSH) exerted a direct stimulatory action on the OSE and induced stem cells proliferation and differentiation into premeiotic germ cell clusters during intact chemoablated ovaries culture. The FSH analog pregnant mare serum gonadotropin treatment to chemoablated mice increased the percentage of surviving VSELs in ovary. The results of this study provide evidence for the presence of potential VSELs in mouse ovaries and show that they survive chemotherapy, are modulated by FSH, and retain the ability to undergo oocyte-specific differentiation. These results show relevance to women who undergo premature ovarian failure because of oncotherapy.
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Affiliation(s)
- Kalpana Sriraman
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (NIRRH), Mumbai, Maharashtra, India
| | - Deepa Bhartiya
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (NIRRH), Mumbai, Maharashtra, India
| | - Sandhya Anand
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (NIRRH), Mumbai, Maharashtra, India
| | - Smita Bhutda
- Stem Cell Biology Department, National Institute for Research in Reproductive Health (NIRRH), Mumbai, Maharashtra, India
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32
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Bukovsky A. Novel methods of treating ovarian infertility in older and POF women, testicular infertility, and other human functional diseases. Reprod Biol Endocrinol 2015; 13:10. [PMID: 25889983 PMCID: PMC4414002 DOI: 10.1186/s12958-015-0001-8] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/10/2014] [Accepted: 01/28/2015] [Indexed: 12/11/2022] Open
Abstract
In vitro maturation (IVM) and in vitro fertilization (IVF) technologies are facing with growing demands of older women to conceive. Although ovarian stem cells (OSCs) of older women are capable of producing in vitro fresh oocyte-like cells (OLCs), such cells cannot respond to IVM and IVF due to the lack of granulosa cells required for their maturation. Follicular renewal is also dependent on support of circulating blood mononuclear cells. They induce intermediary stages of meiosis (metaphase I chromosomal duplication and crossover, anaphase, telophase, and cytokinesis) in newly emerging ovarian germ cells, as for the first time demonstrated here, induce formation of granulosa cells, and stimulate follicular growth and development. A pretreatment of OSC culture with mononuclear cells collected from blood of a young healthy fertile woman may cause differentiation of bipotential OSCs into both developing germ and granulosa cells. A small blood volume replacement may enable treatment of ovarian infertility in vivo. The transferred mononuclear cells may temporarily rejuvenate virtually all tissues, including improvement of the function of endocrine tissues. Formation of new follicles and their development may be sufficient for IVM and IVF. The novel proposed in vitro approaches may be used as a second possibility. Infertility of human males affects almost a half of the infertility cases worldwide. Small blood volume replacement from young healthy fertile men may also be easy approach for the improvement of sperm quality in older or other affected men. In addition, body rejuvenation by small blood volume replacement from young healthy individuals of the same sex could represent a decline of in vitro methodology in favor of in vivo treatment for human functional diseases. Here we propose for the first time that blood mononuclear cells are essential for rejuvenation of those tissues, where immune system components participate in an appropriate division and differentiation of tissue stem cells. If needed, small blood volume replacement from distinct young healthy individuals could be utilized in six month intervals for repair of young altered or aged reproductive and other tissue functions. Systemic and local use of honey bee propolis tincture is an alternative option for functional rejuvenation of some tissues.
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Affiliation(s)
- Antonin Bukovsky
- The Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
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Lai D, Xu M, Zhang Q, Chen Y, Li T, Wang Q, Gao Y, Wei C. Identification and characterization of epithelial cells derived from human ovarian follicular fluid. Stem Cell Res Ther 2015; 6:13. [PMID: 25889077 PMCID: PMC4392788 DOI: 10.1186/s13287-015-0004-6] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2014] [Revised: 02/03/2015] [Accepted: 02/03/2015] [Indexed: 12/13/2022] Open
Abstract
Introduction Follicular fluid is important for follicular development and oocyte maturation. Evidence suggests that follicular fluid is not only rich in proteins but cells. Besides oocytes, the follicular fluid contains granulosa, thecal, and ovarian surface epithelial cells, and both granulosa and thecal cells are well-characterized. However, epithelial cells in follicular fluid are poorly studied. This study aims to isolate and characterize in vitro epithelial cells that originate from human ovarian follicular fluid retrieved in the assisted fertilization program. Methods Follicular fluid samples were collected from 20 women in the assisted reproduction program. Epithelial cells were characterized by flow cytometry assay, immunofluorescence staining, real-time PCR, and time lapse photography. Results Epithelial cell cultures were established from 18 samples. A small population of epithelial cells expresses germ-line stem cell markers, such as octamer-binding transcription factor 4 (OCT4), NANOG, and DEAD box polypeptide 4 (DDX4). In the epithelial cell culture system, oocyte-like cells formed spontaneously in vitro and expressed the following transcription markers: deleted in azoospermia-like (DAZL), developmental pluripotency associated protein 3 stella-related protein (STELLA), zona pellucida gene family C (ZPC), Syntaptonemal complex protein (SCP), and growth and differentiation factor 9 (GDF9). Some of the oocyte-like cells developed a zona pellucida-like structure. Both the symmetric and asymmetric division split of epithelial cells and early developing oocytes were observed using time lapse photography. Cell colonies were formed during epithelial culturing, which maintained and proliferated in an undifferentiated way on the feeder layer and expressed some pluripotency markers. These colonies differentiated in vitro into various somatic cell types in all three germ layers, but did not form teratoma when injected into immunodeficient mice. Furthermore, these epithelial cells could be differentiated directly to functional hepatocyte-like cells, which do not exist in ovarian tissues. Conclusions The epithelial cells derived from follicular fluid are a potential stem cell source with a pluripotent/multipotent character for safe application in oogenesis and regenerative medicine. Electronic supplementary material The online version of this article (doi:10.1186/s13287-015-0004-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Dongmei Lai
- The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China.
| | - Minhua Xu
- The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China.
| | - Qiuwan Zhang
- The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China.
| | - Yifei Chen
- The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China.
| | - Ting Li
- The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China.
| | - Qian Wang
- The Center of Research Laboratory, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200030, China.
| | - Yimeng Gao
- State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.
| | - Chunsheng Wei
- Eye and ENT Hospital, Fudan University, Shanghai, 200031, China.
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Bhartiya D, Hinduja I, Patel H, Bhilawadikar R. Making gametes from pluripotent stem cells--a promising role for very small embryonic-like stem cells. Reprod Biol Endocrinol 2014; 12:114. [PMID: 25421462 PMCID: PMC4255929 DOI: 10.1186/1477-7827-12-114] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/30/2014] [Accepted: 09/01/2014] [Indexed: 01/15/2023] Open
Abstract
The urge to have one's own biological child supersedes any desire in life. Several options have been used to obtain gametes including pluripotent stem cells (embryonic ES and induced pluripotent iPS stem cells); gonadal stem cells (spermatogonial SSCs, ovarian OSCs stem cells), bone marrow, mesenchymal cells and fetal skin. However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints. A tremendous leap in the field occurred using mouse ES and iPS cells wherein they were first differentiated into epiblast-like cells and then primordial germ cell-like cells. These on further development produced sperm, oocytes and live offspring (had associated genetic problems). Evidently differentiating pluripotent stem cells into primordial germ cells (PGCs) remains a major bottleneck. Against this backdrop, we propose that a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) may serve as an alternative, potential source of autologus gametes, keeping in mind that they are indeed PGCs surviving in adult mammalian ovaries and testes. Both VSELs and PGCs are pluripotent, relatively quiescent because of epigenetic modifications of parentally imprinted genes loci like Igf2-H19 and KCNQ1p57, share several markers like Stella, Fragilis, Mvh, Dppa2, Dppa4, Sall4, Blimp1 and functional receptors. VSELs are localized in the basement membrane of seminiferous tubules in testis and in the ovary surface epithelium. Ovarian stem cells from mouse, rabbit, sheep, marmoset and humans (menopausal women and those with premature ovarian failure) spontaneously differentiate into oocyte-like structures in vitro with no additional requirement of growth factors. Thus a more pragmatic option to obtain autologus gametes may be the pluripotent VSELs and if we could manipulate them in vivo - existing ethical and epigenetic/genetic concerns associated with in vitro culture may also be minimized. The field of oncofertility may undergo a sea-change and existing strategies of cryopreservation of gametes and gonadal tissue for fertility preservation in cancer patients will necessitate a revision. However, first the scientific community needs to arrive at a consensus about VSELs in the gonads and then work towards exploiting their potential.
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Affiliation(s)
- Deepa Bhartiya
- />Stem Cell Biology Department, National Institute for Research in Reproductive Health (ICMR), Mumbai, 400 012 India
| | - Indira Hinduja
- />Hinduja IVF Centre, PD Hinduja Hospital and Medical Research Centre, Veer Savarkar Marg, Mumbai, 400 016 India
| | - Hiren Patel
- />Stem Cell Biology Department, National Institute for Research in Reproductive Health (ICMR), Mumbai, 400 012 India
| | - Rashmi Bhilawadikar
- />Hinduja IVF Centre, PD Hinduja Hospital and Medical Research Centre, Veer Savarkar Marg, Mumbai, 400 016 India
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Schatten H, Sun QY, Prather R. The impact of mitochondrial function/dysfunction on IVF and new treatment possibilities for infertility. Reprod Biol Endocrinol 2014; 12:111. [PMID: 25421171 PMCID: PMC4297407 DOI: 10.1186/1477-7827-12-111] [Citation(s) in RCA: 112] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/17/2014] [Accepted: 09/04/2014] [Indexed: 11/12/2022] Open
Abstract
Mitochondria play vital roles in oocyte functions and they are critical indicators of oocyte quality which is important for fertilization and development into viable offspring. Quality-compromised oocytes are correlated with infertility, developmental disorders, reduced blastocyst cell number and embryo loss in which mitochondrial dysfunctions play a significant role. Increasingly, women affected by metabolic disorders such as diabetes or obesity and oocyte aging are seeking treatment in IVF clinics to overcome the effects of adverse metabolic conditions on mitochondrial functions and new treatments have become available to restore oocyte quality. The past decade has seen enormous advances in potential therapies to restore oocyte quality and includes dietary components and transfer of mitochondria from cells with mitochondrial integrity into mitochondria-impaired oocytes. New technologies have opened up new possibilities for therapeutic advances which will increase the success rates for IVF of oocytes from women with compromised oocyte quality.
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Affiliation(s)
- Heide Schatten
| | - Qing-Yuan Sun
| | - Randall Prather
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Joo BS, Jung IK, Park MJ, Joo JK, Kim KH, Lee KS. Differential expression of pluripotent and germ cell markers in ovarian surface epithelium according to age in female mice. Reprod Biol Endocrinol 2014; 12:113. [PMID: 25421381 PMCID: PMC4280751 DOI: 10.1186/1477-7827-12-113] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/24/2014] [Accepted: 09/04/2014] [Indexed: 01/17/2023] Open
Abstract
BACKGROUND Many studies have proposed that putative ovarian stem cells (OSCs) derived from the ovarian surface epithelium (OSE) layer of adult mammalian ovaries can produce oocytes. Few studies have reported that ovaries of aged mammalian females including mice and women possess rare premeiotic germ cells that can generate oocytes. However, no studies have reported the changes of OSCs according to the age of the female. Therefore, this study evaluated pluripotent and germ cell marker expression in the intact ovary, scraped OSE, and postcultured OSE according to age in female mice. METHODS C57BL/6 female mice of 2 age groups (6-8 and 28-31 weeks) were superovulated by injection with 5 IU equine chorionic gonadotropin (eCG). Both ovaries were removed after 48 hours and scrapped to obtain OSE. Gene expressions of pluripotent (Oct-4, Sox-2, Nanog) and germ cell markers (c-Kit, GDF-9, and VASA) were evaluated by RT-PCR. VASA and GDF-9 were immune-localized in oocyte-like structures. RESULTS Expressions of germ cell markers in the intact ovary were significantly decreased in aged females, whereas expressions of pluripotent markers were not detected, regardless of age. Scraped OSE expression of all pluripotent and germ cell markers, except for c-Kit, was similar between both age groups. Three weeks postcultured OSE had significantly decreased expression of GDF-9 and VASA , but not c-Kit, in old mice, as compared to young mice; however there was no difference in the expression of other genes. The number of positively stained Oct-4 by immunohistochemistry in postcultured OSE was 2.5 times higher in young mice than aged mice. Oocyte-like structure was spontaneously produced in postcultured OSE. However, while that of young mice revealed a prominent nucleus, zona pellucida-like structure and cytoplasmic organelles, these features were not observed in old mice. CONCLUSIONS These results show that aged female mice have putative OSCs in OSE, but their differentiation potential, as well as the number of OSCs differs from those of young mice.
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Affiliation(s)
- Bo Sun Joo
| | - In Kook Jung
| | - Min Jung Park
| | - Jong Kil Joo
| | - Ki Hyung Kim
| | - Kyu-Sup Lee
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Gheorghisan-Galateanu AA, Hinescu ME, Enciu AM. Ovarian adult stem cells: hope or pitfall? J Ovarian Res 2014; 7:71. [PMID: 25018783 PMCID: PMC4094411 DOI: 10.1186/1757-2215-7-71] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2014] [Accepted: 06/29/2014] [Indexed: 12/22/2022] Open
Abstract
For many years, ovarian biology has been based on the dogma that oocytes reserve in female mammals included a finite number, established before or at birth and it is determined by the number and quality of primordial follicles developed during the neonatal period. The restricted supply of oocytes in adult female mammals has been disputed in recent years by supporters of postnatal neo-oogenesis. Recent experimental data showed that ovarian surface epithelium and cortical tissue from both mouse and human were proved to contain very low proportion of cells able to propagate themselves, but also to generate immature oocytes in vitro or in vivo, when transplanted into immunodeficient mice ovaries. By mentioning several landmarks of ovarian stem cell reserve and addressing the exciting perspective of translation into clinical practice as treatment for infertility pathologies, the purpose of this article is to review the knowledge about adult mammalian ovarian stem cells, a topic that, since the first approach quickly attracted the attention of both the scientific media and patients.
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Affiliation(s)
- Ancuta Augustina Gheorghisan-Galateanu
- Department of Cellular and Molecular Medicine, Carol Davila University of Medicine and Pharmacy, 8 Eroii Sanitari Blvd., 050474 Bucharest, Romania ; C.I.Parhon National Institute of Endocrinology, 8 Eroii Sanitari Blvd., 050474 Bucharest, Romania
| | - Mihail Eugen Hinescu
- Department of Cellular and Molecular Medicine, Carol Davila University of Medicine and Pharmacy, 8 Eroii Sanitari Blvd., 050474 Bucharest, Romania ; V.Babes National Institute of Pathology, 8 Eroii Sanitari Blvd., 050474 Bucharest, Romania
| | - Ana Maria Enciu
- Department of Cellular and Molecular Medicine, Carol Davila University of Medicine and Pharmacy, 8 Eroii Sanitari Blvd., 050474 Bucharest, Romania ; V.Babes National Institute of Pathology, 8 Eroii Sanitari Blvd., 050474 Bucharest, Romania
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Lai D, Wang F, Dong Z, Zhang Q. Skin-derived mesenchymal stem cells help restore function to ovaries in a premature ovarian failure mouse model. PLoS One 2014; 9:e98749. [PMID: 24879098 PMCID: PMC4039525 DOI: 10.1371/journal.pone.0098749] [Citation(s) in RCA: 66] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2014] [Accepted: 05/07/2014] [Indexed: 12/13/2022] Open
Abstract
Skin-derived mesenchymal stem cells (SMSCs) can differentiate into the three embryonic germ layers. For this reason, they are considered a powerful tool for therapeutic cloning and offer new possibilities for tissue therapy. Recent studies showed that skin-derived stem cells can differentiate into cells expressing germ-cell specific markers in vitro and form oocytes in vivo. The idea that SMSCs may be suitable for the treatment of intractable diseases or traumatic tissue damage has attracted attention. To determine the ability of SMSCs to reactivate injured ovaries, a mouse model with ovaries damaged by busulfan and cyclophosphamide was developed and is described here. Female skin-derived mesenchymal stem cells (F-SMSCs) and male skin-derived mesenchymal stem cells (M-SMSCs) from red fluorescence protein (RFP) transgenic adult mice were used to investigate the restorative effects of SMSCs on ovarian function. Significant increases in total body weight and the weight of reproductive organs were observed in the treated animals. Both F-SMSCs and M-SMSCs were shown to be capable of partially restoring fertility in chemotherapy-treated females. Immunostaining with RFP and anti-Müllerian hormone (AMH) antibodies demonstrated that the grafted SMSCs survived, migrated to the recipient ovaries. After SMSCs were administered to the treated mice, real-time PCR showed that the expression levels of pro-inflammatory cytokines TNF-α, TGF-β, IL-8, IL-6, IL-1β, and IFNγ were significantly lower in the ovaries than in the untreated controls. Consistent with this observation, expression of oogenesis marker genes Nobox, Nanos3, and Lhx8 increased in ovaries of SMSCs-treated mice. These findings suggest that SMSCs may play a role within the ovarian follicle microenvironment in restoring the function of damaged ovaries and could be useful in reproductive health.
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Affiliation(s)
- Dongmei Lai
- The Center of Research Laboratory, and Department of Gynecology, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
- * E-mail:
| | - Fangyuan Wang
- The Center of Research Laboratory, and Department of Gynecology, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
| | - Zhangli Dong
- The Center of Research Laboratory, and Department of Gynecology, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
| | - Qiuwan Zhang
- The Center of Research Laboratory, and Department of Gynecology, The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
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Expression of mesenchymal stem cells-related genes and plasticity of aspirated follicular cells obtained from infertile women. BIOMED RESEARCH INTERNATIONAL 2014; 2014:508216. [PMID: 24724084 PMCID: PMC3958784 DOI: 10.1155/2014/508216] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/23/2013] [Revised: 01/21/2014] [Accepted: 01/22/2014] [Indexed: 12/25/2022]
Abstract
After removal of oocytes for in vitro fertilization, follicular aspirates which are rich in somatic follicular cells are discarded in daily medical practice. However, there is some evidence that less differentiated cells with stem cell characteristics are present among aspirated follicular cells (AFCs). The aim of this study was to culture AFCs in vitro and to analyze their gene expression profile. Using the RT2 Profiler PCR array, we investigated the expression profile of 84 genes related to stemness, mesenchymal stem cells (MCSs), and cell differentiation in AFCs enriched by hypoosmotic protocol from follicular aspirates of infertile women involved in assisted reproduction programme in comparison with bone marrow-derived mesenchymal stem cells (BM-MSCs) and fibroblasts. Altogether the expression of 57 genes was detected in AFCs: 16 genes (OCT4, CD49f, CD106, CD146, CD45, CD54, IL10, IL1B, TNF, VEGF, VWF, HDAC1, MITF, RUNX2, PPARG, and PCAF) were upregulated and 20 genes (FGF2, CASP3, CD105, CD13, CD340, CD73, CD90, KDR, PDGFRB, BDNF, COL1A1, IL6, MMP2, NES, NUDT6, BMP6, SMURF2, BMP4, GDF5, and JAG1) were downregulated in AFCs when compared with BM-MSCs. The genes which were upregulated in AFCs were mostly related to MSCs and connected with ovarian function, and differed from those in fibroblasts. The cultured AFCs with predominating granulosa cells were successfully in vitro differentiated into adipogenic-, osteogenic-, and pancreatic-like cells. The upregulation of some MSC-specific genes and in vitro differentiation into other types of cells indicated a subpopulation of AFCs with specific stemness, which was not similar to those of BM-MSCs or fibroblasts.
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Parte S, Bhartiya D, Patel H, Daithankar V, Chauhan A, Zaveri K, Hinduja I. Dynamics associated with spontaneous differentiation of ovarian stem cells in vitro. J Ovarian Res 2014; 7:25. [PMID: 24568237 PMCID: PMC4234975 DOI: 10.1186/1757-2215-7-25] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2013] [Accepted: 02/17/2014] [Indexed: 01/26/2023] Open
Abstract
BACKGROUND Recent studies suggest that ovarian germ line stem cells replenish oocyte-pool in adult stage, and challenge the central doctrine of 'fixed germ cell pool' in mammalian reproductive biology. Two distinct populations of spherical stem cells with high nucleo-cytoplasmic ratio have been recently identified in the adult mammalian ovary surface epithelium (OSE) including nuclear OCT-4A positive very small embryonic-like (VSELs) and cytoplasmic OCT-4 expressing ovarian germ stem cells (OGSCs). Three weeks culture of scraped OSE cells results in spontaneous differentiation of the stem cells into oocyte-like, parthenote-like, embryoid body-like structures and also embryonic stem cell-like colonies whereas epithelial cells attach and transform into a bed of mesenchymal cells. Present study was undertaken, to further characterize ovarian stem cells and to comprehend better the process of spontaneous differentiation of ovarian stem cells into oocyte-like structures in vitro. METHODS Ovarian stem cells were enriched by immunomagnetic sorting using SSEA-4 as a cell surface marker and were further characterized. Stem cells and clusters of OGSCs (reminiscent of germ cell nests in fetal ovaries), were characterized by immuno-localization for stem and germ cell specific markers and spontaneous differentiation in OSE cultures was studied by live cell imaging. RESULTS Differential expression of markers specific for pluripotent VSELs (nuclear OCT-4A, SSEA-4, CD133), OGSCs (cytoplasmic OCT-4) primordial germ cells (FRAGILIS, STELLA, VASA) and germ cells (DAZL, GDF-9, SCP-3) were studied. Within one week of culture, stem cells became bigger in size, developed abundant cytoplasm, differentiated into germ cells, revealed presence of Balbiani body-like structure (mitochondrial cloud) and exhibited characteristic cytoplasmic streaming. CONCLUSIONS Presence of germ cell nests, Balbiani body-like structures and cytoplasmic streaming extensively described during fetal ovary development, are indeed well recapitulated during in vitro oogenesis in adult OSE cultures along with characteristic expression of stem/germ cell/oocyte markers. Further studies are required to assess the genetic integrity of in vitro derived oocytes before harnessing their clinical potential. Advance in our knowledge about germ cell differentiation from stem cells will enable researchers to design better in vitro strategies which in turn may have relevance to reproductive biology and regenerative medicine.
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Abstract
SummaryFor decades, scientists have considered that female mammals are born with a lifetime reserve of oocytes in the ovary, irrevocably fated to decline after birth. However, controversy in the matter of the possible presence of oocytes and granulosa cells that originate from stem cells in the adult mammalian ovaries has been expanded. The restricted supply of oocytes in adult female mammals has been disputed in recent years by supporters of neo-oogenesis, who claim that germline stem cells (GSCs) exist in the ovarian surface epithelium (OSE) or the bone marrow (BM). Differentiation of ovarian stem cells (OSCs) into oocytes, fibroblast-like cells, granulosa phenotype, neural and mesenchymal type cells and generation of germ cells from OSCs under the contribution of an OSC niche that consists of immune system-related cells and hormonal signalling has been claimed. Although these arguments have met with intense suspicion, their confirmation would necessitate the revision of the current classic knowledge of female reproductive biology.
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Bai Y, Yu M, Hu Y, Qiu P, Liu W, Zheng W, Peng S, Hua J. Location and characterization of female germline stem cells (FGSCs) in juvenile porcine ovary. Cell Prolif 2013; 46:516-28. [PMID: 24033494 DOI: 10.1111/cpr.12058] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2013] [Accepted: 06/28/2013] [Indexed: 12/25/2022] Open
Abstract
OBJECTIVES Existence of germline stem cells (GSCs) in juvenile mammalian female ovaries has been drastically debated recently since reports that adult mouse ovaries still have mitotically active germ cells have been proposed. In addition, definitive location of such female germline stem cells (FGSCs) had not been demonstrated. MATERIALS AND METHODS We segregated porcine FGSCs mechanically from ovary cortex, and tested our hypotheses by utilizing immunofluorescent staining, qRT-PCR and western blotting. RESULTS We attached emphasis to unambiguous location of FGSCs, which settle simultaneously in the theca. Dissected cells from porcine thecal layers maintained similar characteristics to mouse FGSCs and ESCs over 4-months in vitro culture. CONCLUSION These results may provide a new resource for the study of oogenesis and therapy for ovarian sterility.
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Affiliation(s)
- Y Bai
- College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering and Technology, Key Lab for Animal Biotechnology of Agriculture Ministry of China, Northwest A&F University, Yangling, Shaanxi, 712100, China
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Abd-Allah SH, Shalaby SM, Pasha HF, El-Shal AS, Raafat N, Shabrawy SM, Awad HA, Amer MG, Gharib MA, El Gendy EA, Raslan AA, El-Kelawy HM. Mechanistic action of mesenchymal stem cell injection in the treatment of chemically induced ovarian failure in rabbits. Cytotherapy 2013; 15:64-75. [PMID: 23260087 DOI: 10.1016/j.jcyt.2012.08.001] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2012] [Accepted: 08/16/2012] [Indexed: 01/02/2023]
Abstract
BACKGROUND No curative treatment is known for primary ovarian failure; however, mesenchymal stem cells (MSCs), through self-renewal and regeneration, push the trial to evaluate their role in the treatment of ovarian failure. The aim of this study was to explore the impact of MSCs on cyclophosphamide (CTX)-induced ovarian failure in rabbits and to clarify the mechanism(s) by which MSCs exert their action. METHODS Thirty-five adult female rabbits were injected with CTX to induce ovarian failure. Five rabbits were euthanized after the last injection of CTX for histological examination. The others (30 rabbits) were further subdivided into two groups: group 1 (ovarian failure group, 15 rabbits) received no treatment; group 2 (ovarian failure and MSC recipient group, 15 rabbits) received MSCs isolated from extracted bone marrow of male rabbits. RESULTS A decrease of follicle-stimulating hormone and an increase of estrogen and vascular endothelial growth factor (VEGF) levels in the MSC recipient group versus the ovarian failure group were found. Weak caspase-3 expression and +ve proliferating cell nuclear antigen staining after MSC injection were detected. Cytological and histological examinations showed increased follicle numbers with apparent normal structure of ovarian follicles in the MSC recipient group. Moreover, Y chromosome-containing cells from male donors were present within the ovarian tissues in group 2. CONCLUSIONS The current study suggests that intravenous injection of MSCs into rabbits with chemotherapy-induced ovarian damage improved ovarian function. MSCs accomplish this function by direct differentiation into specific cellular phenotypes and by secretion of VEGF, which influence the regeneration of the ovary.
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Affiliation(s)
- Somia H Abd-Allah
- Medical Biochemistry Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt.
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Dzafic E, Stimpfel M, Virant-Klun I. Plasticity of granulosa cells: on the crossroad of stemness and transdifferentiation potential. J Assist Reprod Genet 2013; 30:1255-61. [PMID: 23893266 DOI: 10.1007/s10815-013-0068-0] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2013] [Accepted: 07/19/2013] [Indexed: 01/13/2023] Open
Abstract
The ovarian follicle represents the basic functional unit of the ovary and consists of an oocyte, which is surrounded by granulosa cells (GCs). GCs play an important role in the growth and development of the follicle. They are subject to increased attention since it has recently been shown that the subpopulation of GCs within the growing follicle possesses exceptionally plasticity showing stem cell characteristics. In assisted reproduction programs, oocytes are retrieved from patients together with GCs, which are currently discarded daily, but could be an interesting subject to be researched and potentially used in regenerative medicine in the future. Isolated GCs expressed stem cell markers such as OCT-4, NANOG and SOX-2, showed high telomerase activity, and were in vitro differentiated into other cell types, otherwise not present within ovarian follicles. Recently another phenomenon demonstrated in GCs is transdifferentiation, which could explain many ovarian pathological conditions. Possible applications in regenerative medicine are also given.
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Affiliation(s)
- Edo Dzafic
- Department of Obstetrics and Gynaecology, University Medical Centre Ljubljana, Šlajmerjeva 3, 1000, Ljubljana, Slovenia
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Pathways by which the interplay of organismic and environmental factors lead to phenotypic variation within and across generations. ADVANCES IN CHILD DEVELOPMENT AND BEHAVIOR 2013; 44:325-54. [PMID: 23834011 DOI: 10.1016/b978-0-12-397947-6.00012-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
The range of responses made to environmental exigencies by animals, including humans, may be impacted by the experiences of their progenitors. In mammals, pathways have been documented ranging from transactions between a mother and her developing fetus in the womb through continuity of parenting practices and cultural inheritance. In addition, phenotypic plasticity may be constrained by factors transmitted by the gametes that are involved in the regulation of gene expression rather than modifications to the genome itself. Possible mediators for this kind of inheritance are examined, and the conditions that might have led to the evolution of such transmission are considered. Anticipatory adjustments to possible environmental exigencies are likely to occur when such conditions recur regularly, but intermittently across generations and endure for substantial periods of time, and when adjusting to them after the fact is likely to be biologically costly, even life-threatening. It appears that physical growth and responses to nutrient availability are domains in which anticipatory, epigenetically inherited adjustments occur. In addition, given the fact that humans have oppressed one another repeatedly and for relatively long periods of time, such behavioral tendencies as boldness or innovativeness may be behavioral traits subject to such effects. The implications of these factors for research and policy are discussed.
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Isolation of small SSEA-4-positive putative stem cells from the ovarian surface epithelium of adult human ovaries by two different methods. BIOMED RESEARCH INTERNATIONAL 2013; 2013:690415. [PMID: 23509763 PMCID: PMC3590614 DOI: 10.1155/2013/690415] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/13/2012] [Accepted: 12/27/2012] [Indexed: 12/02/2022]
Abstract
The adult ovarian surface epithelium has already been proposed as a source of stem cells and germinal cells in the literature, therefore it has been termed the “germinal epithelium”. At present more studies have confirmed the presence of stem cells expressing markers of pluripotency in adult mammalian ovaries, including humans. The aim of this study was to isolate a population of stem cells, based on the expression of pluripotency-related stage-specific embryonic antigen-4 (SSEA-4) from adult human ovarian surface epithelium by two different methods: magnetic-activated cell sorting and fluorescence-activated cell sorting. Both methods made it possible to isolate a similar, relatively homogenous population of small, SSEA-4-positive cells with diameters of up to 4 μm from the suspension of cells retrieved by brushing of the ovarian cortex biopsies in reproductive-age and postmenopausal women and in women with premature ovarian failure. The immunocytochemistry and genetic analyses revealed that these small cells—putative stem cells—expressed some primordial germ cell and pluripotency-related markers and might be related to the in vitro development of oocyte-like cells expressing some oocyte-specific transcription factors in the presence of donated follicular fluid with substances important for oocyte growth and development. The stemness of these cells needs to be further researched.
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Celik O, Celik E, Turkcuoglu I, Yilmaz E, Simsek Y, Tiras B. Germline cells in ovarian surface epithelium of mammalians: a promising notion. Reprod Biol Endocrinol 2012; 10:112. [PMID: 23245287 PMCID: PMC3566967 DOI: 10.1186/1477-7827-10-112] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/21/2012] [Accepted: 12/14/2012] [Indexed: 01/03/2023] Open
Abstract
It is a long held doctrine in reproductive biology that women are born with a finite number of oocytes and there is no oogenesis during the postnatal period. However, recent evidence challenges this by showing the presence of germ line stem cells in the human ovarian surface epithelium (OSE), which can serve as a source of germ cells, and differentiate into oocyte like structures. Postnatal renewal of oocytes may have enormous therapeutic potential especially in women facing the risk of premature ovarian failure idiopathically or iatrogenically after exposure to gonadotoxic chemotherapy and radiation for cancer therapy.This article reviews current knowledge on germ line stem cells in human OSE.
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Affiliation(s)
- Onder Celik
- Department of Obstetrics and Gynecology, Inonu University, Medical Faculty, Malatya, Turkey
| | - Ebru Celik
- Department of Obstetrics and Gynecology, Inonu University, Medical Faculty, Malatya, Turkey
| | - Ilgin Turkcuoglu
- Department of Obstetrics and Gynecology, Inonu University, Medical Faculty, Malatya, Turkey
| | - Ercan Yilmaz
- Department of Obstetrics and Gynecology, Inonu University, Medical Faculty, Malatya, Turkey
| | - Yavuz Simsek
- Department of Obstetrics and Gynecology, Inonu University, Medical Faculty, Malatya, Turkey
| | - Bulent Tiras
- Department of Obstetric and Gynecology, Acibadem University, School of Medicine, Istanbul, Turkey
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Bukovsky A, Caudle MR. Immunoregulation of follicular renewal, selection, POF, and menopause in vivo, vs. neo-oogenesis in vitro, POF and ovarian infertility treatment, and a clinical trial. Reprod Biol Endocrinol 2012; 10:97. [PMID: 23176151 PMCID: PMC3551781 DOI: 10.1186/1477-7827-10-97] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/23/2012] [Accepted: 11/11/2012] [Indexed: 12/13/2022] Open
Abstract
The immune system plays an important role in the regulation of tissue homeostasis ("tissue immune physiology"). Function of distinct tissues during adulthood, including the ovary, requires (1) Renewal from stem cells, (2) Preservation of tissue-specific cells in a proper differentiated state, which differs among distinct tissues, and (3) Regulation of tissue quantity. Such morphostasis can be executed by the tissue control system, consisting of immune system-related components, vascular pericytes, and autonomic innervation. Morphostasis is established epigenetically, during morphogenetic (developmental) immune adaptation, i.e., during the critical developmental period. Subsequently, the tissues are maintained in a state of differentiation reached during the adaptation by a "stop effect" of resident and self renewing monocyte-derived cells. The later normal tissue is programmed to emerge (e.g., late emergence of ovarian granulosa cells), the earlier its function ceases. Alteration of certain tissue differentiation during the critical developmental period causes persistent alteration of that tissue function, including premature ovarian failure (POF) and primary amenorrhea. In fetal and adult human ovaries the ovarian surface epithelium cells called ovarian stem cells (OSC) are bipotent stem cells for the formation of ovarian germ and granulosa cells. Recently termed oogonial stem cells are, in reality, not stem but already germ cells which have the ability to divide. Immune system-related cells and molecules accompany asymmetric division of OSC resulting in the emergence of secondary germ cells, symmetric division, and migration of secondary germ cells, formation of new granulosa cells and fetal and adult primordial follicles (follicular renewal), and selection and growth of primary/preantral, and dominant follicles. The number of selected follicles during each ovarian cycle is determined by autonomic innervation. Morphostasis is altered with advancing age, due to degenerative changes of the immune system. This causes cessation of oocyte and follicular renewal at 38 +/-2 years of age due to the lack of formation of new granulosa cells. Oocytes in primordial follicles persisting after the end of the prime reproductive period accumulate genetic alterations resulting in an exponentially growing incidence of fetal trisomies and other genetic abnormalities with advanced maternal age. The secondary germ cells also develop in the OSC cultures derived from POF and aging ovaries. In vitro conditions are free of immune mechanisms, which prevent neo-oogenesis in vivo. Such germ cells are capable of differentiating in vitro into functional oocytes. This may provide fresh oocytes and genetically related children to women lacking the ability to produce their own follicular oocytes. Further study of "immune physiology" may help us to better understand ovarian physiology and pathology, including ovarian infertility caused by POF or by a lack of ovarian follicles with functional oocytes in aging ovaries. The observations indicating involvement of immunoregulation in physiological neo-oogenesis and follicular renewal from OSC during the fetal and prime reproductive periods are reviewed as well as immune system and age-independent neo-oogenesis and oocyte maturation in OSC cultures, perimenopausal alteration of homeostasis causing disorders of many tissues, and the first OSC culture clinical trial.
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Affiliation(s)
- Antonin Bukovsky
- The Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
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Bhartiya D, Sriraman K, Gunjal P, Modak H. Gonadotropin treatment augments postnatal oogenesis and primordial follicle assembly in adult mouse ovaries? J Ovarian Res 2012; 5:32. [PMID: 23134576 PMCID: PMC3616927 DOI: 10.1186/1757-2215-5-32] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2012] [Accepted: 10/13/2012] [Indexed: 01/07/2023] Open
Abstract
BACKGROUND Follicle stimulating hormone (FSH) exerts action on both germline and somatic compartment in both ovary and testis although FSH receptors (FSHR) are localized only on the somatic cells namely granulosa cells of growing follicles and Sertoli cells in the seminiferous tubules. High levels of FSH in females are associated with poor ovarian reserve, ovarian hyper stimulation syndrome etc. and at the same time FSH acts as a survival factor during in vitro organotypic culture of ovarian cortical strips. Thus a further understanding of FSH action on the ovary is essential. We have earlier reported presence of pluripotent very small embryonic-like stem cells (VSELs express Oct-4A in addition to other pluripotent markers) and their immediate descendants 'progenitors' ovarian germ stem cells (OGSCs express Oct-4B in addition to other germ cell markers) in ovarian surface epithelium (OSE) in various mammalian species including mice, rabbit, monkey, sheep and human. Present study was undertaken to investigate the effect of pregnant mare serum gonadotropin (PMSG) on adult mice ovaries with a focus on VSELs, OGSCs, postnatal oogenesis and primordial follicle assembly. METHODS Ovaries were collected from adult mice during different stages of estrus cycle and after 2 and 7 days of PMSG (5 IU) treatment to study histo-architecture and expression for FSHR, pluripotent stem cells , meiosis and germ cell specific markers. RESULTS PMSG treatment resulted in increased FSHR and proliferation as indicated by increased FSHR and PCNA immunostaining in OSE and oocytes of primordial follicles (PF) besides the granulosa cells of large antral follicles. Small 1-2 regions of multilayered OSE invariably associated with a cohort of PF during estrus stage in control ovary were increased to 5-8 regions after PMSG treatment. This was associated with an increase in pluripotent transcripts (Oct-4A, Nanog), meiosis (Scp-3) and germ cells (Oct-4B, Mvh) specific markers. MVH showed positive immuno staining on germ cell nest-like clusters and at places primordial follicles appeared connected through oocytes. CONCLUSIONS The results of the present study show that gonadotropin (PMSG) treatment to adult mouse leads to increased pluripotent stem cell activity in the ovaries, associated with increased meiosis, appearance of several cohorts of PF and their assembly in close proximity of OSE. This was found associated with the presence of germ cell nests and cytoplasmic continuity of oocytes in PF. We have earlier reported that pluripotent ovarian stem cells in the adult mammalian ovary are the VSELs which give rise to slightly differentiated OGSCs. Thus we propose that gonadotropin through its action on pluripotent VSELs augments neo-oogenesis and PF assembly in adult mouse ovaries.
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Affiliation(s)
- Deepa Bhartiya
- Stem Cell Biology Department, National Institute for Research in Reproductive Health, Mumbai, 400 012, India.
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